BPC-1: a secreted brain-specific protein expressed and secreted by prostate and bladder cancer cells

ABSTRACT

Described is a novel gene and its encoded secreted tumor antigen, termed BPC-1, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers which express BPC-1, particularly including prostate cancer and bladder cancer. In human normal tissues, BPC-1 is only expressed in certain tissues of the brain. However, BPC-1 is expressed at high levels in prostate cancer cells and is also expressed in bladder cancer cells. The structure of BPC-1 includes a signal sequence and a CUB domain. BPC-1 protein is secreted. Preliminary experimental evidence suggests that BPC-1 is directly involved in oncogenesis or maintenance of the transformed phenotype of cancer cells expressing BPC-1. BPC-1 also appears to bind specifically to a cellular protein expressed in prostate cancer cells and other cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/061,341, filed 18 Feb. 2005, now U.S. Pat. No. 7,785,811, issued 31Aug. 2010, which is a continuation of U.S. patent application Ser. No.09/887,593, filed 21 Jun. 2001, now abandoned, which is a divisional ofU.S. patent application Ser. No. 09/374,135, filed 10 Aug. 1999, nowU.S. Pat. No. 6,277,972, issued 21 Aug. 2001, which claims the benefitof U.S. Provisional Application No. 60/095,982, filed 10 Aug. 1998. Thecontents of the applications listed in this paragraph are fullyincorporated by reference herein.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference inits entirety for all purposes. The sequence listing is identified on theelectronically filed text file as follows:

File Name Date of Creation Size (bytes) 511582001802Seqlist.txt Feb. 17,2010 13,634 bytes

TECHNICAL FIELD

The invention described herein relates to a novel gene and its encodedsecreted tumor antigen, termed BPC-1, and to diagnostic and therapeuticmethods and compositions useful in the management of various cancerswhich express BPC-1, particularly including prostate cancer and bladdercancer.

BACKGROUND ART

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people each year, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Many cancer patients experience a recurrence.

Generally speaking, the fundamental problem in the management of thedeadliest cancers is the lack of effective and non-toxic systemictherapies. Molecular medicine, still very much in its infancy, promisesto redefine the ways in which these cancers are managed. Unquestionably,there is an intensive worldwide effort aimed at the development of novelmolecular approaches to cancer diagnosis and treatment. For example,there is a great interest in identifying truly tumor-specific genes andproteins that could be used as diagnostic and prognostic markers and/ortherapeutic targets or agents. Research efforts in these areas areencouraging, and the increasing availability of useful moleculartechnologies has accelerated the acquisition of meaningful knowledgeabout cancer. Nevertheless, progress is slow and generally uneven.

As discussed below, the management of prostate cancer serves as a goodexample of the limited extent to which molecular biology has translatedinto real progress in the clinic. With limited exceptions, the situationis more or less the same for the other major carcinomas mentioned above.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most common malecancer and is the second leading cause of cancer death in men. In theUnited States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, and chemotherapy remain fixed as the main treatment modalities.Unfortunately, these treatments are ineffective for many and are oftenassociated with significant undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the management of this disease. Although the serum PSAassay has been a very useful tool, its specificity and general utilityis widely regarded as lacking in several important respects, as furtherdiscussed below. Most prostate cancers initially occur in the peripheralzone of the prostate gland, away from the urethra. Tumors within thiszone may not produce any symptoms and, as a result, most men withearly-stage prostate cancer will not present clinical symptoms of thedisease until significant progression has occurred. Tumor progressioninto the transition zone of the prostate may lead to urethralobstruction, thus producing the first symptoms of the disease. However,these clinical symptoms are indistinguishable from the commonnon-malignant condition of benign prostatic hyperplasia (BPH). Earlydetection and diagnosis of prostate cancer currently relies on digitalrectal examinations (ORE), prostate specific antigen (PSA) measurements,transrectal ultrasonography (TRUS), and trans rectal needle biopsy(TRNB). At present, serum PSA measurement in combination with ORErepresent the leading tool used to detect and diagnose prostate cancer.Both have major limitations which have fueled intensive research intofinding better diagnostic markers of this disease.

Similarly, there is no available marker that can predict the emergenceof the typically fatal metastatic stage of prostate cancer. Diagnosis ofmetastatic stage is presently achieved by open surgical or laparoscopicpelvic lymphadenectomy, whole body radionuclide scans, skeletalradiography, and/or bone lesion biopsy analysis. Clearly, better imagingand other less invasive diagnostic methods offer the promise of easingthe difficulty those procedures place on a patient, as well as improvingdiagnostic accuracy and opening therapeutic options. A similar problemis the lack of an effective prognostic marker for determining whichcancers are indolent and which ones are or will be aggressive. PSA, forexample, fails to discriminate accurately between indolent andaggressive cancers. Until there are prostate tumor markers capable ofreliably identifying early-stage disease, predicting susceptibility tometastasis, and precisely imaging tumors, the management of prostatecancer will continue to be extremely difficult.

PSA is the most widely used tumor marker for screening, diagnosis, andmonitoring prostate cancer today. In particular, several immunoassaysfor the detection of serum PSA are in widespread clinical use. Recently,a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for PSAmRNA in serum has been developed. However, PSA is not a disease-specificmarker, as elevated levels of PSA are detectable in a large percentageof patients with BPH and prostatitis (25-86%) (Gao et al., 1997,Prostate 31:264-281), as well as in other nonmalignant disorders and insome normal men, a factor which significantly limits the diagnosticspecificity of this marker. For example, elevations in serum PSA ofbetween 4 to 10 ng/ml are observed in BPH, and even higher values areobserved in prostatitis, particularly acute prostatitis. BPH is anextremely common condition in men. Further confusing the situation isthe fact that serum PSA elevations may be observed without anyindication of disease from ORE, and visa-versa. Moreover, it is nowrecognized that PSA is not prostate-specific (Gao et al., supra, forreview).

Various methods designed to improve the specificity of PSA-baseddetection have been described, such as measuring PSA density and theratio of free vs. complexed PSA. However, none of these methodologieshave been able to reproducibly distinguish benign from malignantprostate disease. In addition, PSA diagnostics have sensitivities ofbetween 57-79% (Cupp & Osterling, 1993, Mayo Clin Proc 68:297-306), andthus miss identifying prostate cancer in a significant population of menwith the disease.

There are some known markers which are expressed predominantly inprostate, such as prostate specific membrane antigen (PSM), a hydrolasewith 85% identity to a rat neuropeptidase (Carter et al., 1996, Proc.Natl. Acad. Sci. USA 93:749; Bzdega et al., 1997, J. Neurochem. 69:2270). However, the expression of PSM in small intestine and brain(Israeli et al., 1994, Cancer Res. 54: 1807), as well its potential rolein neuropeptide catabolism in brain, raises concern of potentialneurotoxicity with anti-PSM therapies. Preliminary results using anIndium-111 labeled, anti-PSM monoclonal antibody to image recurrentprostate cancer show some promise (Sodee et al., 1996, Clin Nuc Med21:759-766). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93:7252) andprostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl.Acad. Sci. USA 95:1735). PCTA-1, a novel galectin, is largely secretedinto the media of expressing cells and may hold promise as a diagnosticserum marker for prostate cancer (Su et al., 1996). PSCA, a GPI-linkedcell surface molecule, was cloned from LAPC-4 cDNA and is unique in thatit is expressed primarily in basal cells of normal prostate tissue andin cancer epithelia (Reiter et al., 1998). Vaccines for prostate cancerare also being actively explored with a variety of antigens, includingPSM and PSA.

DISCLOSURE OF THE INVENTION

The present invention relates to a novel secreted protein designatedBPC-1. In normal individuals, BPC-1 protein is only expressed in certaintissues of the brain. In prostate cancer, BPC-1 is expressed at highlevels in tumor cells. BPC-1 is also expressed in bladder cancer cells,and may be expressed in other cancer cells. The structure of BPC-1includes a signal sequence and a CUB domain. The BPC-1 protein CUBdomain is structurally similar to the CUB domains of several otherproteins.

The BPC-1 gene therefore encodes a secreted tumor antigen which may beuseful as a diagnostic, staging and/or prognostic marker, and/or mayserve as an excellent target for various approaches to the treatment ofprostate, bladder and other cancers expressing BPC-1. Although theprecise function of BPC-1 is presently unknown, preliminary experimentalevidence suggests that BPC-1 is directly involved in oncogenesis ormaintenance of the transformed phenotype of cancer cells expressingBPC-1. BPC-1 also appears to bind specifically to a cellular proteinexpressed in prostate cancer cells and other cells. Taken together, thisevidence indicates that BPC-1 is functionally involved in an oncogenicpathway. As further described herein, this understanding leads to anumber of potential approaches to the treatment of cancers expressingBPC-1 involving the inhibition of BPC-1 function.

The invention provides polynucleotides corresponding or complementary toall or part of the BPC-1 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding BPC-1proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and relatedmolecules, polynucleotides or oligonucleotides complementary to theBPC-1 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides which hybridize to the BPC-1 genes, mRNAs, or toBPC-1-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding BPC-1. Recombinant DNA molecules containingBPC-1 polynucleotides, cells transformed or transduced with suchmolecules, and host-vector systems for the expression of BPC-1 geneproducts are also provided. The invention further provides BPC-1proteins and polypeptide fragments thereof. The invention furtherprovides antibodies that bind to BPC-1 proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, antibodies labeled with a detectable marker,and antibodies conjugated to radionuclides, toxins or other therapeuticcompositions. The invention further provides methods for detecting thepresence of BPC-1 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express a BPC-1.The invention further provides various therapeutic compositions andstrategies for treating cancers which express BPC-1 such as prostate andbladder cancers, including antibody, vaccine and small molecule therapy,and therapies aimed at inhibiting the transcription, translation,processing or function of BPC-1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A-C). Molecular structure of human BPC-1: Nucleotide and deducedamino acid sequences of BPC-1 clone 6 cDNA (SEQ ID NOS: 1 and 2,respectively). The signal sequence is indicated in boldface, the CUBdomain in underlined boldface, and the SSH-derived nucleic acid sequencein boldface.

FIG. 2. Molecular structure of human BPC-1: Schematic representation ofthe human BPC-1 structure. Percentage CG contents across regions of thesequence are also indicated.

FIG. 3. Molecular structure of human BPC-1: Amino acid sequencealignment of the BPC-1 CUB domain with CUB domains from various knownproteins. (A) Alignment of BPC-1 with C. elegans CUB domain protein (SEQID NO: 3; Wilson et al., 1994, Nature 368:32-38), and (B) alignmentswith the CUB domains of murine BMP-1 (SEQ ID NO: 4-8; Fukagawa et al.1994, Dev. Biol 163:175-183). Percent sequence identities are indicatedon the figure.

FIG. 4. Northern blot analysis of human BPC-1 expression in variousnormal tissues showing exclusive expression in brain.

FIG. 5. Semi-quantitative RT-PCR expression analysis showing human BPC-1expression in prostate cancer xenografts (lanes 3-5 of panel A) and alimited number of normal human tissues (lanes 1-2 of panel A; lanes 1-8of panels B and C).

FIG. 6. RNA dot blot analysis of human BPC-1 mRNA expression in 37normal tissues, showing expression only in specific regions of thebrain.

FIG. 7. Northern blot analysis of human BPC-1 mRNA expression incortical regions of the brain, showing expression only in specificregions of the cortex.

FIG. 8. Semi-quantitative RT-PCR expression analysis of human BPC-1expression in fetal tissues, showing that BPC-1 expression ispredominant in fetal brain, and is also expressed at lower levels in anumber of other fetal tissues.

FIG. 9. Northern blot analysis of human BPC-1 mRNA expression in a panelof prostate and bladder carcinoma xenografts and/or cell lines, showingexpression in all prostate cancer xenograft samples, the LnCAP prostatecancer cell line, and a bladder carcinoma cell line.

FIG. 10. SDS-PAGE autoradiography of immunoprecipitated recombinanthuman BPC-1 protein secreted into the tissue culture media of BPC-1transfected 293T cells.

FIG. 11. Western blot analysis of recombinant human BPC-1 protein, asexpressed in HighFive insect cells infected with a BPC-1 encodingbaculovirus, showing processed mature BPC-1 and unprocessed precursorBPC-1 in cell Iysates and low levels of processed mature BPC-1 in cellmedia.

FIG. 12. Northern blot analysis of recombinant human BPC-1 expressed byPC3 and 3T3CL7 cells infected with BPC-1 encoding retrovirus.

FIG. 13. Western blot detection of BPC-1 protein in tissue culturesupernatants of cells expressing the BPC-1 gene. 25 μl of neatsupernatant from of various cell lines was subjected to Western blotanalysis using a 1:500 dilution of murine anti-BPC-1 polyclonal serum.The blot was then incubated with anti-mouse-HRP conjugated secondaryantibody and BPC-1 specific signals were visualized by enhancedchemiluminescent detection. Left blot. Lane 1: Affinity (nickel)purified MYC/HIS BPC-1; lane 2: 293T control cells, 24 hr conditionedmedium. Right blot. Lane 1: 293T control cells, 24 hr conditionedmedium; Lane 2: 293T transfected with a MYC/HIS tagged BPC1 vector, 24hr conditioned medium; Lane 3: 293T cells transfected with an alkalinephosphatase (AP)/BPC-1 fusion vector, 24 hr conditioned medium; Lane 4:PC3 cells infected with control Neo retrovirus and G418 selected, 4 dayconditioned medium; Lane 5: PC3 cells infected with BPC-1 retrovirus andG418 selected, 4 day conditioned medium stored 1 week at 4C; Lane 6: PC3cells infected with BPC-1 retrovirus and G418 selected, 4 dayconditioned medium; Lane 7: NIH3T3 cells acutely infected with controlNeo retrovirus, 4 day conditioned medium; Lane 8: NIH3T3 cells infectedwith BPC1 retrovirus and G418 selected, 4 day conditioned medium; Lane8: NIH3T3 cells acutely infected with BPC1 retrovirus, 4 day conditionedmedium.

FIG. 14. Western blot analysis showing that BPC-1-AP is present inconditioned media. The lanes contain 20 μl conditioned media from 293Tcells or 293T cells collected 48 hours after media change fromtransfections with the BPC-1-AP construct or with a construct having APalone. Anti-HIS antibodies were used to detect proteins.

FIG. 15. BPC-1-AP binds to a 45 kDa protein using a far-westernanalysis. Lysates from brain, testis, prostate, the xenografts LAPC4ADand LAPC9AD, and the cell lines 3T3, LAPC4, LNCaP, and PC-3 were used tomake the western blots. The blots were incubated with conditioned mediafrom a 293T cell line producing only secreted alkaline phosphatase (B)and with media containing BPC-1-AP (A). The alkaline phosphatase signalswere detected using a chemiluminescent AP detection system.

MODES OF CARRYING OUT THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers which have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1-C2 disease under the Whitmore-Jewettsystem, and stage T3-T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers which have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is the preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months thereafter. The most common site forprostate cancer metastasis is bone. Prostate cancer bone metastases are,on balance, characteristically osteoblastic rather than osteolytic(i.e., resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

As used herein, the term “polypeptide” means a polymer of at least 10amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37 degrees C. and temperatures for washingin 0.1×SSC/0.1% SDS are above 55 degrees C., and most preferably tostringent hybridization conditions.

In the context of amino acid sequence comparisons, the term “identity”is used to express the percentage of amino acid residues at the samerelative position which are the same. Also in this context, the term“homology” is used to express the percentage of amino acid residues atthe same relative positions which are either identical or are similar,using the conserved amino acid criteria of BLAST analysis, as isgenerally understood in the art. Further details regarding amino acidsubstitutions, which are considered conservative under such criteria,are provided below.

Additional definitions are provided throughout the subsections whichfollow.

Molecular and Biochemical Features of BPC-1

As is further described in the Examples which follow, the BPC-1 gene andprotein have been characterized using a number of analytical approaches.For example, analyses of nucleotide coding and amino acid sequences wereconducted in order to identify potentially related molecules, as well asrecognizable structural domains, topological features, and otherelements within the BPC-1 mRNA and protein structure. RT-PCR andNorthern blot analyses of BPC-1 mRNA expression were conducted in orderto establish the range of normal and cancerous tissues expressing BPC-1message. Western blot analyses of BPC-1 protein expression inexperimentally transfected cells were conducted to determine celllocalization and secretion of processed and unprocessed recombinanthuman BPC-1 protein. Functional assays designed to determine BPC-1interaction with cellular binding partner(s) and activity were alsoconducted.

BPC-1 is an oncogenic, secreted, CUB domain-containing protein which isexpressed in prostate and bladder carcinoma cells and binds to acellular protein. BPC-1 expression is exquisitely brain-specific innormal adult human tissues. In fetal tissues, BPC-1 expression ispredominant in brain, but is also turned on in a number of otherdeveloping organs and tissues. BPC-1 gene expression is activated inhuman prostate cancer. In particular, BPC-1 is expressed at very highlevels in androgen dependent human prostate tumor xenografts originallyderived from a patient with high grade metastatic prostate cancer, andis expressed at lower but significant levels in other prostate cancersamples. BPC-1 is also expressed at high levels in at least some bladdercarcinomas.

The BPC-1 protein is initially translated into a 158 amino acidprecursor containing a signal sequence. During post-translationalprocessing, the signal sequence is cleaved to yield the mature 135 aminoacid secreted protein. The 5′ non-coding region of the BPC-1 gene isextremely G/C rich (approximately 72% G/C content, compared to 42% inthe coding region and 30% in the 3′ non-coding region), implying thatthis region of the gene contains elements involved in transcriptional ortranslational control (FIG. 1).

The BPC-1 primary structure contains a recognizable CUB domain(Complement subcomponents C1r/C1s, Uegf, Bmp1) (Borck and Beckmann,1993, J. Mol. Biol. 231:539-545) which shares homology with other CUBdomain proteins (FIG. 1; FIG. 3). CUB domains were originally found incomplement subcomponents C1r and C1s, and were subsequently identifiedin Uegf (epidermal growth factor related sea urchin protein) and Bmp1(bone morphogenetic protein 1), a protease involved in bone development.Functionally, CUB domains have been associated with protein interaction,receptor binding and other activities. Unlike other CUB domain proteinswhich have additional enzymatic functions, BPC-1 is unique in that it isessentially a secreted CUB domain with no other apparent functionaldomains. The CUB domain of BPC-1 could function as a protein-proteininteraction domain, mediating interactions with other secretedmolecules, extracellular matrix molecules and/or cell surface receptors.This would imply a potential growth-factor or cell stimulator function.

The presence of a CUB domain in the BPC-1 structure further supports theconclusion that BPC-1 interacts with and probably binds to otherproteins. The CUB domain, viewed as an extracellular domain involved inprotein-protein interaction, occurs in many diverse secreted or cellsurface proteins involved in a variety developmental processes (Borckand Beckmann, 1993, J. Mol. Biol. 231: 539-545). One family of proteinscharacterized by CUB domains, to which BPC-1 protein may bear somerelation, are the Spermadhesins. The Spermadhesins are CUB domaincontaining secreted proteins produced by the seminal vesicles and areestimated to be about 15-18 kd in size (approx. 140 amino acids); theseproteins function to inhibit sperm motility and are inactivated byproteolysis (Iwamoto et al., 1995, FEBBS Letters 368:420-424).

Preliminary experimental evidence suggests that BPC-1 is directlyinvolved in oncogenesis or maintenance of the transformed phenotype ofcancer cells expressing BPC-1. In this regard, BPC-1 shows transformingactivity in soft agar assays and binds to a cellular protein expressedby cells including those expressing BPC-1. Taken together, this evidenceindicates that BPC-1 is functionally involved in an oncogenic pathway,and that BPC-1 activity in this pathway may occur through interactionwith a BPC-1 binding partner or through binding to or association withother protein(s). As further described herein, this understanding leadsto a number of potential approaches to the treatment of cancersexpressing BPC-1, involving the inhibition of BPC-1 function.

BPC-1 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a BPC-1 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a BPC-1 protein and fragments thereof, DNA, RNA, DNA/RNAhybrid, and related molecules, polynucleotides or oligonucleotidescomplementary to a BPC-1 gene or mRNA sequence or a part thereof, andpolynucleotides or oligonucleotides which hybridize to a BPC-1 gene,mRNA, or to a BPC-1-encoding polynucleotide (collectively, “BPC-1polynucleotides”). As used herein, the BPC-1 gene and protein is meantto include the BPC-1 gene and protein specifically described herein andthe genes and proteins corresponding to other BPC-1 proteins andstructurally similar variants of the foregoing. Such other BPC-1proteins and variants will generally have coding sequences which arehighly homologous to the BPC-1 and/or BPC1-2 coding sequences, andpreferably will share at least about 50% amino acid identity and atleast about 60% amino acid homology (using BLAST criteria), morepreferably sharing 70% or greater homology (using BLAST criteria).

A BPC-1 polynucleotide may comprise a polynucleotide having thenucleotide sequence of human BPC-1 as shown in FIG. 1, a sequencecomplementary to the foregoing, or a polynucleotide fragment of any ofthe foregoing. Another embodiment comprises a polynucleotide which iscapable of hybridizing under stringent hybridization conditions to thehuman BPC-1 cDNA shown in FIG. 1 or to a polynucleotide fragmentthereof.

Specifically contemplated are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone or including alternative bases, whether derivedfrom natural sources or synthesized. For example, antisense moleculescan be RNAs or other molecules, including peptide nucleic acids (PNAs)or non-nucleic acid molecules, such as phosphorothioate derivatives,that specifically bind DNA or RNA in a base pair-dependent manner. Askilled artisan can readily obtain these classes of nucleic acidmolecules using the BPC-1 polynucleotides and polynucleotide sequencesdisclosed herein.

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of thepolynucleotides of the invention or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of a BPC-1 polynucleotide in a sample and as ameans for detecting a cell expressing a BPC-1 protein. Examples of suchprobes include polypeptides comprising all or part of the human BPC-1cDNA sequence shown in FIG. 1. Examples of primer pairs capable ofspecifically amplifying BPC-1 mRNAs are also described in the Exampleswhich follow. As will be understood by the skilled artisan, a great manydifferent primers and probes may be prepared based on the sequencesprovided in herein and used effectively to amplify and/or detect a BPC-1mRNA.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides whichcorrespond or are complementary to genes other than the BPC-1 gene orwhich encode polypeptides other than BPC-1 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated BPC-1 polynucleotide.

The BPC-1 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the BPC-1 gene(s), mRNA(s), orfragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of BPC-1 polypeptides; as tools for modulatingor inhibiting the expression of the BPC-1 gene(s) and/or translation ofthe BPC-1 transcript(s); and as therapeutic agents.

Methods for Isolating BPC-1-Encoding Nucleic Acid Molecules

The BPC-1 cDNA sequences described herein enable the isolation of otherpolynucleotides encoding BPC-1 gene product(s), as well as the isolationof polynucleotides encoding BPC-1 gene product homologues, alternativelyspliced isoforms, allelic variants, and mutant forms of the BPC-1 geneproduct. Various molecular cloning methods that can be employed toisolate full length cDNAs encoding a BPC-1 gene are well known (See, forexample, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2dedition., Cold Spring Harbor Press, New York, 1989; Current Protocols inMolecular Biology. Ausubel et al., eds., Wiley and Sons, 1995). Forexample, lambda phage cloning methodologies may be convenientlyemployed, using commercially available cloning systems (e.g., Lambda ZAPExpress, Stratagene). Phage clones containing BPC-1 gene cDNAs may beidentified by probing with a labeled BPC-1 cDNA or a fragment thereof.For example, in one embodiment, the BPC-1 cDNA (FIG. 1) or a portionthereof can be synthesized and used as a probe to retrieve overlappingand full length cDNAs corresponding to a BPC-1 gene. The BPC-1 geneitself may be isolated by screening genomic DNA libraries, bacterialartificial chromosome libraries (BACs), yeast artificial chromosomelibraries (YACs), and the like, with BPC-1 DNA probes or primers.

Recombinant DNA Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga BPC-1 polynucleotide, including but not limited to phages, plasmids,phagemids, cosmids, YACs, BACs, as well as various viral and non-viralvectors well known in the art, and cells transformed or transfected withsuch recombinant DNA or RNA molecules. As used herein, a recombinant DNAor RNA molecule is a DNA or RNA molecule that has been subjected tomolecular manipulation in vitro. Methods for generating such moleculesare well known (see, for example, Sambrook et al., 1989, supra).

The Invention further provides a host-vector system comprising arecombinant DNA molecule containing a BPC-1 polynucleotide within asuitable prokaryotic or eukaryotic host cell. Examples of suitableeukaryotic host cells include a yeast cell, a plant cell, or an animalcell, such as a mammalian cell or an insect cell (e.g., abaculovirus-infectible cell such as an Sf9 or HghFive cell). Examples ofsuitable mammalian cells include various prostate cancer cell lines suchLnCaP, PC-3, DU145, LAPC-4, TsuPr1, other transfectable or transducibleprostate cancer cell lines, as well as a number of mammalian cellsroutinely used for the expression of recombinant proteins (e.g., COS,CHO, 293, 293T cells). More particularly, a polynucleotide comprisingthe coding sequence of a BPC-1 may be used to generate BPC-1 proteins orfragments thereof using any number of host-vector systems routinely usedand widely known in the art.

A wide range of host-vector systems suitable for the expression of BPC-1proteins or fragments thereof are available, see for example, Sambrooket al., 1989, supra; Current Protocols in Molecular Biology, 1995,supra). Preferred vectors for mammalian expression include but are notlimited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vectorpSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expressionvectors, BPC-1 may be preferably expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1,3T3, PC-3, LNCaP and TsuPr1. The host-vector systems of the inventionare useful for the production of a BPC-1 protein or fragment thereof.Such host-vector systems may be employed to study the functionalproperties of BPC-1 and BPC-1 mutations.

Mature recombinant human BPC-1 protein may be produced and secreted bymammalian cells transfected with a construct encoding precursor BPC-1.In a particular embodiment described in the Examples, 293T cells aretransfected with an expression plasmid encoding the precursor form ofBPC-1 (i.e., including the signal sequence) and mature BPC-1 protein issecreted into the cell culture medium where it may be convenientlyisolated using standard purification methods. Mature recombinant humanBPC-1 may also be produced by cells which process but do not secrete themature protein. One example of such a system is a BPC-1 encodingbaculovirus-infected cell. As described in the examples, such cellsexpress and process high levels of BPC-1 intracellularly. The matureBPC-1 protein may be recovered, in such cases, from cell lysates usingstandard procedures. Whether the mature BPC-1 is secreted or is retainedintracellularly by the host cell, BPC-1 may be affinity purified frommedia or cell lysates using BPC-1 antibodies.

Proteins encoded by the BPC-1 genes, or by fragments thereof, will havea variety of uses, including but not limited to generating antibodiesand in methods for identifying ligands and other agents and cellularconstituents that bind to a BPC-1 gene product. Antibodies raisedagainst a BPC-1 protein or fragment thereof may be useful in diagnosticand prognostic assays, imaging methodologies (including, particularly,cancer imaging), and therapeutic methods in the management of humancancers characterized by expression of a BPC-1 protein, including butnot limited to cancer of the prostate. Various immunological assaysuseful for the detection of BPC-1 proteins are contemplated, includingbut not limited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), immunocytochemical methods, and the like. Such antibodies maybe labeled and used as immunological imaging reagents capable ofdetecting prostate cells (e.g., in radioscintigraphic imaging methods).BPC-1 proteins may also be particularly useful in generating cancervaccines, as further described below.

BPC-1 Proteins

Another aspect of the present invention provides BPC-1 proteins andpolypeptide fragments thereof. The BPC-1 proteins of the inventioninclude those specifically identified herein, as well as allelicvariants, conservative substitution variants and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined below. Fusion proteins which combineparts of different BPC-1 proteins or fragments thereof, as well asfusion proteins of a BPC-1 protein and a heterologous polypeptide arealso included. Such BPC-1 proteins will be collectively referred to asthe BPC-1 proteins, the proteins of the invention, or BPC-1. As usedherein, the term “BPC-1 polypeptide” refers to a polypeptide fragment ora BPC-1 protein of at least 10 amino acids, preferably at least 15 aminoacids.

A specific embodiment of a BPC-1 protein comprises a polypeptide havingthe amino acid sequence of human BPC-1 as shown in FIG. 1, from aboutamino acid residue number 1 through about amino acid residue number 158as shown therein. Another specific embodiment of a BPC-1 proteincomprises a polypeptide having the amino acid sequence of human BPC-1 asshown in FIG. 1, from about amino acid residue number 24 through aboutamino acid residue number 158 as shown therein.

In general, naturally occurring allelic variants of human BPC-1 willshare a high degree of structural identity and homology (e.g., 90% ormore identity). Typically, allelic variants of the BPC-1 proteins willcontain conservative amino acid substitutions within the BPC-1 sequencesdescribed herein or will contain a substitution of an amino acid from acorresponding position in a BPC-1 homologue. One class of BPC-1 allelicvariants will be proteins that share a high degree of homology with atleast a small region of a particular BPC-1 amino acid sequence, but willfurther contain a radical departure form the sequence, such as anon-conservative substitution, truncation, insertion or frame shift.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

BPC-1 proteins may be embodied in many forms, preferably in isolatedform. As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the BPC-1 proteinfrom cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated BPC-1 protein. A purified BPC-1 proteinmolecule will be substantially free of other proteins or molecules whichimpair the binding of BPC-1 to antibody or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a BPC-1 protein include a purified BPC-1 protein and afunctional, soluble BPC-1 protein. In one form, such functional, solubleBPC-1 proteins or fragments thereof retain the ability to bind antibodyor other ligand.

The invention also provides BPC-1 polypeptides comprising biologicallyactive fragments of the BPC-1 amino acid sequence, such as a polypeptidecorresponding to part of the amino acid sequences for BPC-1 as shown inFIG. 1. Such polypeptides of the invention exhibit properties of theBPC-1 protein, such as the ability to elicit the generation ofantibodies which specifically bind an epitope associated with the BPC-1protein.

BPC-1 polypeptides can be generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the artbased on the amino acid sequences of the human BPC-1 proteins disclosedherein. Alternatively, recombinant methods can be used to generatenucleic acid molecules that encode a polypeptide fragment of a BPC-1protein. In this regard, the BPC-1-encoding nucleic acid moleculesdescribed herein provide means for generating defined fragments of BPC-1proteins. BPC-1 polypeptides are particularly useful in generating andcharacterizing domain specific antibodies (e.g., antibodies recognizingan extracellular or intracellular epitope of a BPC-1 protein), inidentifying agents or cellular factors that bind to BPC-1 or aparticular structural domain thereof, and in various therapeuticcontexts, including but not limited to cancer vaccines. BPC-1polypeptides containing particularly interesting structures can bepredicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-BPC-1 antibodies or in identifying cellular factors thatbind to BPC-1.

In a specific embodiment described in the examples which follow, maturesecreted BPC-1 is conveniently expressed in 293T cells transfected witha CMV-driven expression vector encoding BPC-1 with a C-terminal 6×Hisand MYC tag (pcDNA3.1/mycHIS. Invitrogen). The secreted HIS-tagged BPC-1in the culture media may be purified using a nickel column usingstandard techniques.

BPC-1 Antibodies

Another aspect of the invention provides antibodies that bind to BPC-1proteins and polypeptides. The most preferred antibodies willselectively bind to a BPC-1 protein and will not bind (or will bindweakly) to non-BPC-1 proteins and polypeptides. Anti-BPC-1 antibodiesthat are particularly contemplated include monoclonal and polyclonalantibodies as well as fragments containing the antigen binding domainand/or one or more complementarity determining regions of theseantibodies. As used herein, an antibody fragment is defined as at leasta portion of the variable region of the immunoglobulin molecule whichbinds to its target, i.e., the antigen binding region.

BPC-1 antibodies of the invention may be particularly useful in prostatecancer therapeutic strategies, diagnostic and prognostic assays, andimaging methodologies. Similarly, such antibodies may be useful in thetreatment, diagnosis, and/or prognosis of other cancers, to the extentBPC-1 is also expressed or overexpressed in other types of cancer. Onesuch cancer that expresses BPC-1 is bladder carcinoma.

The invention also provides various immunological assays useful for thedetection and quantification of BPC-1 and mutant BPC-1 proteins andpolypeptides. Such assays generally comprise one or more BPC-1antibodies capable of recognizing and binding a BPC-1 or mutant BPC-1protein, as appropriate, and may be performed within variousimmunological assay formats well known in the art, including but notlimited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), and the like. In addition, immunological imaging methodscapable of detecting prostate cancer are also provided by the invention,including but limited to radioscintigraphic imaging methods usinglabeled BPC-1 antibodies. Such assays may be clinically useful in thedetection, monitoring, and prognosis of prostate cancer, particularlyadvanced prostate cancer.

BPC-1 antibodies may also be used in methods for purifying BPC-1 andmutant BPC-1 proteins and polypeptides and for isolating BPC-1homologues and related molecules. For example, in one embodiment, themethod of purifying a BPC-1 protein comprises incubating a BPC-1antibody, which has been coupled to a solid matrix, with a lysate orother solution containing BPC-1 under conditions which permit the BPC-1antibody to bind to BPC-1; washing the solid matrix to eliminateimpurities; and eluting the BPC-1 from the coupled antibody. Other usesof the BPC-1 antibodies of the invention include generatingantiidiotypic antibodies that mimic the BPC-1 protein.

BPC-1 antibodies may also be used therapeutically by, for example,modulating or inhibiting the biological activity of a BPC-1 protein ortargeting and destroying cancer cells expressing a BPC-1 protein orBPC-1 binding partner. Because BPC-1 is a secreted protein which appearsto bind to a cellular protein and because BPC-1 appears to haveoncogenic activity, antibodies may be therapeutically useful forblocking BPC-1's ability to bind to its receptor or interact with otherproteins through which it exerts its oncogenic biological activity. In aparticular embodiment, a BPC-1 specific antibody or combination thereof(preferably a monoclonal antibody or combination thereof) isadministered to a patient suffering from a BPC-1 expressing tumor suchthat the antibody binds to BPC-1 and inhibits its ability to execute itsfunction. BPC-1 antibody therapy is more specifically described in theTherapeutic Methods and Compositions subsection below.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a BPC-1 protein, peptide, or fragment, in isolatedor immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press,Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring HarborPress, NY (1989)). In addition, fusion proteins of BPC-1 may also beused, such as a BPC-1 GST-fusion protein. In a particular embodiment, aGST fusion protein comprising all or most of the open reading frameamino acid sequence of FIG. 1 may be produced and used as an immunogento generate appropriate antibodies. Cells expressing or overexpressingBPC-1 may also be used for immunizations. Similarly, any cell engineeredto express BPC-1 may be used. Such strategies may result in theproduction of monoclonal antibodies with enhanced capacities forrecognizing endogenous BPC-1. Another useful immunogen comprises BPC-1proteins linked to the plasma membrane of sheep red blood cells. Inaddition, naked DNA immunization techniques known in the art may be used(with or without purified BPC-1 protein or BPC-1 expressing cells) togenerate an immune response to the encoded immunogen (for review, seeDonnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

The amino acid sequence of BPC-1 as shown in FIG. 1 may be used toselect specific regions of the BPC-1 protein for generating antibodies.For example, hydrophobicity and hydrophilicity analyses of the BPC-1amino acid sequence may be used to identify hydrophilic regions in theBPC-1 structure. Regions of the BPC-1 protein that show immunogenicstructure, as well as other regions and domains, can readily beidentified using various other methods known in the art, such asChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis.

Methods for the generation of BPC-1 antibodies are further illustratedby way of the examples provided herein.

Methods for preparing a protein or polypeptide for use as an immunogenand for preparing immunogenic conjugates of a protein with a carriersuch as BSA, KLH, or other carrier proteins are well known in the art.In some circumstances, direct conjugation using, for example,carbodiimide reagents may be used; in other instances linking reagentssuch as those supplied by Pierce Chemical Co., Rockford, Ill., may beeffective. Administration of a BPC-1 immunogen is conducted generally byinjection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

BPC-1 monoclonal antibodies are preferred and may be produced by variousmeans well known in the art. For example, immortalized cell lines whichsecrete a desired monoclonal antibody may be prepared using the standardmethod of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the BPC-1 protein orBPC-1 fragment. When the appropriate immortalized cell culture secretingthe desired antibody is identified, the cells may be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the BPC-1 protein can also be produced in the contextof chimeric or CDR grafted antibodies of multiple species origin.Humanized or human BPC-1 antibodies may also be produced and arepreferred for use in therapeutic contexts. Methods for humanizing murineand other non-human antibodies by substituting one or more of thenon-human antibody CDRs for corresponding human antibody sequences arewell known (see for example, Jones et al., 1986, Nature 321: 522-525;Riechmnan et al, 1988, Nature 332:323-327; Verhoeyen et al., 1988,Science 239:1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad.Sci. USA 89:4285 and Sims et al., 1993, J. Immunol. 151:2296. Methodsfor producing fully human monoclonal antibodies include phage displayand transgenic methods (for review, see Vaughan et al., 1998, NatureBiotechnology 16:535-539).

Fully human BPC-1 monoclonal antibodies may be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display) (Griffiths and Hoogenboom, “Building an in vitroimmune system: human antibodies from phage display libraries,” inProtein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man, Clark, M. (ed.), Nottingham Academic,pp. 45-64 (1993); Burton and Barbas, “Human Antibodies fromCombinatorial Libraries,” id., pp. 65-82). Fully human BPC-1 monoclonalantibodies may also be produced using transgenic mice engineered tocontain human immunoglobulin gene loci as described in PCT PatentApplication WO98/24893, Kucherlapati and Jakobovits et al., publishedDec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs7(4):607-614). This method avoids the in vitro manipulation requiredwith phage display technology and efficiently produces high affinityauthentic human antibodies.

Reactivity of BPC-1 antibodies with a BPC-1 protein may be establishedby a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,BPC-1 proteins, peptides, BPC-1-expressing cells or extracts thereof.

A BPC-1 antibody or fragment thereof of the invention may be labeledwith a detectable marker or conjugated to a second molecule, such as acytotoxic agent, and used for targeting the second molecule to a BPC-1positive cell (Vitetta, E. S. et al., 1993, “Immunotoxin Therapy,” inDeVita, Jr., V. T. et al., eds., Cancer: Principles and Practice ofOncology, 4th ed., J.B. Lippincott Co., Philadelphia, 2624-2636).Examples of cytotoxic agents include, but are not limited to ricin,ricin A-chain, doxorubicin, daunorubicin, taxol, ethiduim bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin D, diphteria toxin, Pseudomonasexotoxin (PE) A, PE40, abrin, arbrin A chain, modeccin A chain,alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin,curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, andglucocorticoid and other chemotherapeutic agents, as well asradioisotopes such as ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. Antibodies may also beconjugated to an anti-cancer pro-drug activating enzyme capable ofconverting the pro-drug to its active form. See, for example, U.S. Pat.No. 4,975,287.

Further, bi-specific antibodies specific for two or more BPC-1 epitopesmay be generated using methods generally known in the art. Further,antibody effector functions may be modified so as to enhance thetherapeutic effect of BPC-1 antibodies on the growth of cancer cells.Homodimeric antibodies may also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53:2560-2565). Suchantibodies may provide a means for achieving enhanced BPC-1 inhibition.

Methods for the Detection of BPC-1

Another aspect of the present invention relates to methods for detectingBPC-1 polynucleotides and BPC-1 proteins, as well as methods foridentifying a cell which expresses BPC-1.

More particularly, the invention provides assays for the detection ofBPC-1 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable BPC-1 polynucleotides include, for example, a BPC-1gene or fragments thereof, BPC-1 mRNA, alternative splice variant BPC-1mRNAs, and recombinant DNA or RNA molecules containing a BPC-1polynucleotide. A number of methods for amplifying and/or detecting thepresence of BPC-1 polynucleotides are well known in the art and may beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a BPC-1 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a BPC-1polynucleotides as sense and antisense primers to amplify BPC-1 cDNAstherein; and detecting the presence of the amplified BPC-1 cDNA. Inanother embodiment, a method of detecting a BPC-1 gene in a biologicalsample comprises first isolating genomic DNA from the sample; amplifyingthe isolated genomic DNA using BPC-1 polynucleotides as sense andantisense primers to amplify the BPC-1 gene therein; and detecting thepresence of the amplified BPC-1 gene. Any number of appropriate senseand antisense probe combinations may be designed from the nucleotidesequences provided for BPC-1 (FIG. 1) and used for this purpose.

The invention also provides assays for detecting the presence of a BPC-1protein in a tissue of other biological sample such as serum, bone,prostate, and other tissues, urine, cell preparations, and the like.Methods for detecting a BPC-1 protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, WesternBlot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, in one embodiment, a method of detecting the presence of aBPC-1 protein in a biological sample comprises first contacting thesample with a BPC-1 antibody, a BPC-1-reactive fragment thereof, or arecombinant protein containing an antigen binding region of a BPC-1antibody; and then detecting the binding of BPC-1 protein in the samplethereto.

Methods for identifying a cell which expresses BPC-1 are also provided.In one embodiment, an assay for identifying a cell which expresses aBPC-1 gene comprises detecting the presence of BPC-1 mRNA in the cell.Methods for the detection of particular mRNAs in cells are well knownand include, for example, hybridization assays using complementary DNAprobes (such as in situ hybridization using labeled BPC-1 riboprobes,Northern blot and related techniques) and various nucleic acidamplification assays (such as RT-PCR using complementary primersspecific for BPC-1, and other amplification type detection methods, suchas, for example, branched DNA, SISBA, TMA and the like). Alternatively,an assay for identifying a cell which expresses a BPC-1 gene comprisesdetecting the presence of BPC-1 protein in the cell or secreted by thecell. Various methods for the detection of proteins are well known inthe art and may be employed for the detection of BPC-1 proteins andBPC-1 expressing cells.

BPC-1 expression analysis may also be useful as a tool for identifyingand evaluating agents which modulate BPC-1 gene expression. For example,BPC-1 expression is significantly upregulated in prostate cancer, andmay also be expressed in other cancers. Identification of a molecule orbiological agent that could inhibit BPC-1 expression or over-expressionin cancer cells may be of therapeutic value. Such an agent may beidentified by using a screen that quantifies BPC-1 expression by RT-PCR,nucleic acid hybridization or antibody binding.

Assays for Determining BPC-1 Expression Status

Determining the status of BPC-1 expression patterns in an individual maybe used to diagnose cancer and may provide prognostic information usefulin defining appropriate therapeutic options. Similarly, the expressionstatus of BPC-1 may provide information useful for predictingsusceptibility to particular disease stages, progression, and/or tumoraggressiveness. The invention provides methods and assays fordetermining BPC-1 expression status and diagnosing cancers which expressBPC-1, such as prostate and bladder cancers.

In one aspect, the invention provides assays useful in determining thepresence of cancer in an individual, such as prostate and bladdercancers, comprising detecting a significant increase in BPC-1 mRNA orprotein expression in a test cell or tissue sample relative toexpression levels in the corresponding normal cell or tissue. Thepresence of BPC-1 mRNA may, for example, be evaluated in tissue samplesof the colon, lung, prostate, pancreas, bladder, breast, ovary, cervix,testis, head and neck, brain, stomach, etc. The presence of significantBPC-1 expression in any of these tissues may be useful to indicate theemergence, presence and/or severity of these cancers, since thecorresponding normal tissues do not express BPC-1 mRNA or express it atlower levels.

In a related embodiment, BPC-1 expression status may be determined atthe protein level rather than at the nucleic acid level. For example,such a method or assay would comprise determining the level of BPC-1protein expressed by cells in a test tissue sample or in serum, semen orurine, and comparing the level so determined to the level of BPC-1expressed in a corresponding normal sample. In one embodiment, thepresence of BPC-1 protein is evaluated, for example, usingimmunohistochemical methods. BPC-1 antibodies or binding partnerscapable of detecting BPC-1 protein expression may be used in a varietyof assay formats well known in the art for this purpose. In anotherembodiment, the presence of secreted BPC-1 protein in serum or urine orother body fluids is examined.

Because BPC-1 is a secreted protein expressed in prostate, bladder, andpossibly other cancers, assays for detecting and quantifying BPC-1 inblood or serum are expected to be useful for the detection, diagnosis,prognosis, and/or staging of a BPC-1 expressing tumor in an individual.For example, BPC-1 is not expressed in normal prostate, but is expressedin prostate and bladder cancers. Accordingly, detection of serum BPC-1may provide an indication of the presence of a prostate or bladdertumor. Diagnosis of prostate or bladder cancer may be made on the basisof this information and/or other information. In respect of prostatecancer, for example, such other information may include serum PSAmeasurements, ORE and/or ultrasonography. Further, the level of BPC-1detected in the serum may provide information useful in staging orprognosis. For example, very high levels of BPC-1 protein in serum maysuggest a larger and/or more aggressive tumor.

The brain-specific expression of BPC-1 in normal tissues is expected toprovide an important advantage of this aspect of the invention, namely,very low to non-existent background levels of circulating BPC-1,resulting in a high correlation between the presence of serum BPC-1protein and the presence of cancer. This advantage is expected to resultfrom the characteristics of the blood-brain barrier, a system of tightjunctions in capillaries of the central nervous system that resists thepassage of cells, pathogens and macromolecules into and out of thesubarachnoid space. Accordingly, BPC-1 expressed in the brain is notexpected to be released into the vascular system. Since no other normaltissue tested has demonstrated significant expression of BPC-1, thepresence of serum BPC-1 would strongly suggest the presence of a BPC-1expressing tumor.

In addition, peripheral blood may be conveniently assayed for thepresence of cancer cells, including but not limited to prostate cancer,using RT-PCR to detect BPC-1 expression. The presence of RT-PCRamplifiable BPC-1 mRNA provides an indication of the presence ofprostate cancer. RT-PCR detection assays for tumor cells in peripheralblood are currently being evaluated for use in the diagnosis andmanagement of a number of human solid tumors. In the prostate cancerfield, these include RT-PCR assays for the detection of cells expressingPSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein etal., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin.Chem. 41: 1687-1688). RT-PCR assays are well known in the art.

A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detectingBPC-1 mRNA or BPC-1 protein in a tissue sample, its presence indicatingsusceptibility to cancer, wherein the degree of BPC-1 mRNA expressionpresent is proportional to the degree of susceptibility. In a specificembodiment, the presence of BPC-1 in prostate tissue is examined, withthe presence of BPC-1 in the sample providing an indication of prostatecancer susceptibility (or the emergence or existence of a prostatetumor). In another specific embodiment, the presence of BPC-1 in bladdertissue is examined, with the presence of BPC-1 in the sample providingan indication of bladder cancer susceptibility (or the emergence orexistence of a bladder tumor). In yet another specific embodiment, thepresence of BPC-1 in serum is examined, with the presence of BPC-1providing an indication of susceptibility to (or presence of) a BPC-1expressing tumor, such as a bladder or prostate tumor. In anotherembodiment, the presence of BPC-1 in urine is examined, with thepresence of BPC-1 therein providing an indication of susceptibility to(or presence of) a BPC-1 expressing bladder tumor.

Yet another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of BPC-1 mRNAor BPC-1 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of BPC-1 mRNA or BPC-1 proteinexpressed in a corresponding normal tissue taken from the sameindividual or a normal tissue reference sample, wherein the degree ofBPC-1 mRNA or BPC-1 protein expression in the tumor sample relative tothe normal sample indicates the degree of aggressiveness. In a specificembodiment, aggressiveness of prostate tumors is evaluated bydetermining the extent to which BPC-1 is expressed in the tumor cells,with higher expression levels indicating more aggressive tumors.

In a related embodiment, serum levels of BPC-1 may be used to provide anindication of the extent and aggressiveness of a BPC-1 expressing tumor,wherein higher levels of serum BPC-1 may suggest a more advanced andmore aggressive tumor. Serum BPC-1 measurements over time would beexpected to provide further information, wherein an increase in BPC-1would be expected to reflect progression and the rate of the increasewould be expected to correlate with aggressiveness. Similarly, a declinein serum BPC-1 would be expected to reflect a slower growing orregressing tumor. The identification of BPC-1 in serum may be useful todetect tumor initiation and early stage disease, particularly sincebackground BPC-1 interference is expected to be minimal to non-existentin view of the BPC-1 brain specific expression profile in normalindividuals. In patients who have undergone surgery or therapy, serumBPC-1 levels would be useful for monitoring treatment response andpotential recurrence. As an alternative or adjunct to serum BPC-1measurements, the presence and levels of BPC-1 secreted in urine may beuseful in relation to bladder cancer.

Methods for detecting and quantifying the expression of BPC-1 mRNA orprotein are described herein and use standard nucleic acid and proteindetection and quantification technologies well known in the art.Standard methods for the detection and quantification of BPC-1 mRNAinclude in situ hybridization using labeled BPC-1 riboprobes, Northernblot and related techniques using BPC-1 polynucleotide probes, RT-PCRanalysis using primers specific for BPC-1, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like. In a specific embodiment, semi-quantitative RTPCR may be usedto detect and quantify BPC-1 mRNA expression as described in theExamples which follow. Any number of primers capable of amplifying BPC-1may be used for this purpose, including but not limited to the variousprimer sets specifically described herein. Standard methods for thedetection and quantification of protein may be used for this purpose. Ina specific embodiment, polyclonal or monoclonal antibodies specificallyreactive with the wild-type BPC-1 protein may be used in animmunohistochemical assay of biopsied tissue.

Assays for Circulating an Excreted BPC-1

The mature BPC-1 is a secreted protein. Tumors which express BPC-1 wouldbe expected to secrete BPC-1 into the vasculature, and/or excreted inurine or semen, where the protein may be detected and quantified usingassays and techniques well known in the molecular diagnostic art.Excreted BPC-1 may also be detectable in urine and semen. Detecting andquantifying the levels of circulating or excreted BPC-1 is expected tohave a number of uses in the diagnosis, staging, and prognosis ofprostate, bladder and other such BPC-1 expressing tumors. A number ofdifferent technical approaches for the detection and quantification ofserum proteins are well known in the art.

Detecting BPC-1 protein in urine may indicate the presence of a bladdercancer secreting BPC-1. Normally, significant levels of protein are notdetected in urine, provided that renal function is normal. However,proteins expressed and secreted by bladder cancer cells may enter urinein the bladder directly, permitting their detection in urine.Interestingly, the BPC-1 protein exhibits a relatively high degree ofstability in recombinant cell culture media, suggesting that the proteinmay also remain stable in urine.

In one embodiment, a capture ELISA is used to detect and quantify BPC-1in serum, urine or semen. A capture ELISA for BPC-1 comprises,generally, at least two monoclonal antibodies of different isotypes thatrecognize distinct epitopes of the BPC-1 protein, or one anti-BPC-1monoclonal antibody and a specific polyclonal serum derived from adifferent species (e.g., rabbit, goat, sheep, hamster, etc.). In thisassay, one reagent serves as the capture (or coating) antibody and theother as the detection antibody (see Example 13 herein).

Therapeutic Methods and Compositions

The identification of BPC-1 as a secreted protein which is onlyexpressed in tissues of the brain in normal individuals but which ishighly expressed in prostate cancer (as well as expressed in bladdercarcinoma and possibly other cancers) opens a number of therapeuticapproaches to the treatment of prostate, bladder and potentially othercancers. Applicants' initial functional research suggests that BPC-1 hastransformation activity and that this activity is initiated through theinteraction of BPC-1 to a cellular protein, or through binding to orassociation with another protein. The protein's CUB domain may alsofunction as a protein-protein interaction domain, mediating interactionswith other secreted molecules, extracellular matrix molecules and/orcell surface receptors.

Accordingly, therapeutic approaches aimed at inhibiting the activity ofthe BPC-1 protein are expected to be useful for patients suffering fromprostate cancer, bladder cancer, and other cancers expressing BPC-1.These therapeutic approaches generally fall into two classes. One classcomprises various methods for inhibiting the binding of the BPC-1protein to its receptor, or inhibiting its binding to or associationwith another protein. Another class comprises a variety of methods forinhibiting the transcription of the BPC-1 gene or translation of BPC-1mRNA.

A. Therapeutic Methods Based on Inhibition of BPC-1 Protein Function

Within the first class of therapeutic approaches, the invention includesvarious methods and compositions for inhibiting the binding of BPC-1 toits receptor or other binding partner or its association with otherprotein(s) as well as methods for inhibiting BPC-1 function.

A.1. Therapeutic Inhibition of BPC-1 with BPC-1 Antibodies

In one approach, antibodies which bind to BPC-1 and thereby inhibit theability of BPC-1 to bind to its coordinate binding partner, or to bindto or associate with other protein(s), may be used to attenuate anoncogenic/transformation signal pathway involving BPC-1.

To the extent BPC-1 is involved in initiating, promoting and/orsustaining tumor cell growth or other tumor cell properties through abinding partner-mediated signal, such antibodies are expected to betherapeutically useful.

BPC-1 antibodies and fragments thereof which are capable of inhibitingBPC-1 function are expected to be useful in treating prostate, bladder,and possibly other cancers. Such antibodies may function to inhibitBPC-1 activity in different ways. For example, a BPC-1 antibody mayprevent BPC-1 binding to its receptor or binding to/associating withanother protein.

Alternatively, a BPC-1 antibody may bind to a biologically active domainof the BPC-1 protein, thereby inhibiting function. In this regard,antibodies specifically directed to the BPC-1 CUB domain (see FIG. 1)may be particularly effective in either inhibiting BPC-1 binding (if theCUB domain is functionally involved in binding) or in otherwiseinhibiting the CUB domain's function. Such domain-specific BPC-1antibodies may be generated as previously described. For example, theCUB domain amino acid sequence shown in FIG. 1 may be used to generate aCUB-domain immunogen for the generation of such antibodies.

With respect to the treatment of cancer with BPC-1 antibodies, a numberof factors may be considered, including but not limited to thefollowing. First, monoclonal antibodies are generally preferred,particularly those with very high binding affinity for the secretedBPC-1 protein. Second, fully human or humanized monoclonal antibodiesexhibiting low or no antigenicity in the patient are preferred. The useof murine or other non-human monoclonal antibodies and human/mousechimeric mAbs may induce moderate to strong immune responses in somepatients. Third, the method by which the antibodies are delivered to thepatient may vary with the type of cancer being treated.

Generally, where the therapeutic objective is the inhibition of BPC-1activity or signal transduction in the target tumor tissue,administration of BPC-1 antibodies directly to the tumor site mayprovide local elimination of BPC-1 function sufficient to generate aclinical response. Direct administration of BPC-1 Mabs is also possibleand may have advantages in certain contexts. For example, for thetreatment of bladder carcinoma, BPC-1 Mabs may be injected directly intothe bladder.

Alternatively, BPC-1 antibodies may be administered systemically, whichmay result in elimination of BPC-1 function in the primary tumor, incirculating micrometastasis, and/or in established metastasis. Thedegree of tumor vascularization may provide guidance on which deliveryapproach is recommended. Similarly, the grade and/or stage of diseasewould be expected to provide useful information in this regard. Forexample, a higher grade, more advanced tumor may be more likely to seedmetastasis, suggesting systemic administration in order to treat orprevent the emergence of metastases.

BPC-1 mAbs may be therapeutically useful either alone or as well ascombinations, or “cocktails”, of different mAbs such as thoserecognizing different epitopes. Such mAb cocktails may have certainadvantages inasmuch as they contain mAbs which bind to differentepitopes and enhance the functional inhibition of BPC-1. In addition,the administration of BPC-1 mAbs may be combined with other therapeuticagents, including but not limited to various chemotherapeutic agents,androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF).

Treatment of cancer with a BPC-1 antibody will generally involve theadministration of the BPC-1 antibody preparation via an acceptable routeof administration such as intravenous injection (IV) or bolus infusion,typically at a dose in the range of about 0.1 to about 200 mg/kg bodyweight. Doses in the range of 10-500 mg mAb per week (or more) may beeffective and well tolerated. An initial loading dose followed bysmaller weekly doses of the mAb preparation may be used. As one of skillin the art will understand, various factors will influence the idealdose regimen in a particular case. Such factors may include, forexample, the binding affinity and half life of the mAb or mAbs used, thedegree of BPC-1 expression in the patient, the desired steady-stateantibody concentration level, frequency of treatment, and the influenceof chemotherapeutic agents or other therapies used in combination withthe therapeutic composition.

A.2. Therapeutic Inhibition of BPC-1 with Intracellular Antibodies

In another approach, recombinant vectors encoding single chainantibodies which specifically bind to BPC-1 may be introduced into BPC-1expressing cells via gene transfer technologies, wherein the encodedsingle chain anti-BPC-1 antibody is expressed intracellularly, binds toBPC-1 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, may bespecifically targeted to a particular compartment within the cell,providing a great deal of control over where the inhibitory activity ofthe treatment will be focused. This technology has been successfullyapplied in the art (for review, see Richardson and Marasco, 1995,TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate theexpression of otherwise abundant cell surface receptors. See, forexample, Richardson et al., 1995, Proc. Natl. Acad. Sci. USA92:3137-3141; Beerli et al., 1994, J. Biol. Chem. 289:23931-23936;Deshane et al., 1994, Gene Ther. 1:332-337.

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and is expressed asa single polypeptide. Optionally, single chain antibodies may beexpressed as a single chain variable region fragment joined to the lightchain constant region. Well known intracellular trafficking signals maybe engineered into recombinant polynucleotide vectors encoding suchsingle chain antibodies in order to precisely target the expressedintrabody to the desired intracellular compartment. For example,intrabodies targeted to the endoplasmic reticulum (ER) may be engineeredto incorporate a leader peptide and, optionally, a C-terminal ERretention signal, such as the KDEL amino acid motif. Intrabodiesintended to exert activity in the nucleus may be engineered to include anuclear localization signal. Lipid moieties may be joined to intrabodiesin order to tether the intrabody to the cytosolic side of the plasmamembrane. Intrabodies may also be targeted to exert function in thecytosol. For example, cytosolic intrabodies may be used to sequesterfactors within the cytosol, thereby preventing them from beingtransported to their natural cellular destination.

In one embodiment, intrabodies may be used to capture BPC-1 in the ER,thereby preventing its maturation and secretion outside of the cell.ER-targeting signals and/or leader peptides may be engineered into suchBPC-1 intrabodies in order to achieve the desired targeting. Suchintrabodies would be expected to capture BPC-1 as it is being processedby the ER, thereby inhibiting BPC-1 processing or transport through theplasma membrane of the cell. This method would essentially prevent theexistence of secreted mature bioactive BPC-1 at the level of the ER.Such BPC-1 intrabodies may be designed to bind specifically to aparticular BPC-1 domain, including the signal sequence of precursorBPC-1. Endoplasmic reticulum-targeted intrabodies reactive with theBPC-1 protein would be expected to capture BPC-1 in the endoplasmicreticulum. In another embodiment, intrabodies specifically reactive withthe BPC-1 CUB domain may be used to block BPC-1 CUB domain functionwithin the cytosol.

A.3 Therapeutic Inhibition of BPC-1 with Recombinant Proteins

In another approach, recombinant molecules which are capable of bindingto BPC-1, thereby preventing BPC-1 from accessing/binding to itscoordinate receptor or associating with another protein involved intransmitting an oncogenic signal, may be used to inhibit BPC-1 function.Such recombinant molecules may, for example, contain the reactivepart(s) of a BPC-1 specific antibody molecule. In a particularembodiment, the BPC-1 ligand binding domain of a BPC-1 receptor orbinding partner may be engineered into a dimeric fusion proteincomprising two BPC-1 ligand binding domains linked to the Fc portion ofa human IgG, such as human IgGl. Such IgG portion may contain, forexample, the CH2 and CH3 domains and the hinge region, but not the CH 1domain. Such dimeric fusion proteins may be administered in soluble formto patients suffering from a cancer associated with the expression ofBPC-1, including but not limited to prostate and bladder cancers, wherethe dimeric fusion protein specifically binds to BPC-1, thereby blockingBPC-1 interaction with its receptor or other binding partner. Suchdimeric fusion proteins may be further combined into multimeric proteinsusing known antibody linking technologies.

B. Therapeutic Methods Based on Inhibition of BPC-1 Transcription orTranslation

Within the second class of therapeutic approaches, the inventionprovides various methods and compositions for inhibiting thetranscription of the BPC-1 gene. Similarly, the invention also providesmethods and compositions for inhibiting the translation of BPC-1 mRNAinto protein.

In one approach, a method of inhibiting the transcription of the BPC-1gene comprises contacting the BPC-1 gene with a BPC-1 antisensepolynucleotide. In another approach, a method of inhibiting BPC-1 mRNAtranslation comprises contacting the BPC-1 mRNA with an antisensepolynucleotide. In another approach, a BPC-1 specific ribozyme may beused to cleave the BPC-1 message, thereby inhibiting translation. Suchantisense and ribozyme based methods may also be directed to theregulatory regions of the BPC-1 gene, such as the BPC-1 promoter and/orenhancer elements. Similarly, proteins capable of inhibiting a BPC-1gene transcription factor may be used to inhibit BPC-1 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

The 5′ untranslated region (UTR) of the BPC-1 cDNA of FIG. 1 is anextremely GC rich sequence, strongly implying the presence oftranslational control elements within this part of the BPC-1 mRNA. Thischaracteristic of the BPC-1 gene suggests that blocking accessibility ofthe 5′ UTR may result in inhibition of BPC-1 translation. In oneapproach, an antisense molecule complementary to the 5′ UTR of the BPC-1mRNA is contacted with the 5′ UTR of the BPC-1 message, therebyresulting in hybridization which prevents endogenous BPC-1 translationfactors from accessing the necessary activation element(s) in the BPC-15′ UTR. A modification of this approach uses an polynucleotidecomprising a sequence complementary to the 5′ UTR BPC-1 mRNA joined to aribozyme or similarly active polynucleotide capable of splicing theBPC-1 mRNA, thereby adding a second layer of translational inhibition.

Although, in the above method, a BPC-1 splicing ribozyme may becontacted with the BPC-1 mRNA separately, i.e., not joined to aheterologous sequence such as the anti-5′ UTR mentioned above,delivering the ribozyme or similarly active polynucleotide as part ofsuch a heterologous BPC-1 hybridizing polynucleotide would be expectedto result in placing the ribozyme in direct proximity to the targetsequence and may result in a higher level of inhibitory activity.

Other factors which inhibit the transcription of BPC-1 throughinterfering with BPC-1 transcriptional activation may also be useful forthe treatment of cancers expressing BPC-1, and cancer treatment methodsutilizing such factors are also within the scope of the invention.Similarly, factors which are capable of interfering with BPC-1processing may be useful for the treatment of cancers expressing BPC-1.

C. General Considerations

Gene transfer and gene therapy technologies may be used for deliveringtherapeutic polynucleotide molecules to tumor cells synthesizing BPC-1(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother BPC-1 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding BPC-1 antisensepolynucleotides, ribozymes, factors capable of interfering with BPC-1transcription, factors which are capable of interfering with processingand/or secretion of mature BPC-1, and so forth, may be delivered totarget tumor cells using such gene therapy approaches.

The above therapeutic approaches may be combined with chemotherapy orradiation therapy regimens. These therapeutic approaches may also enablethe use of reduced dosages of concomitant chemotherapy, particularly inpatients that do not tolerate the toxicity of the chemotherapeutic agentwell.

The anti-tumor activity of a particular composition (e.g., antibody,ribozyme, recombinant fusion protein), or a combination of suchcompositions, may be evaluated using various in vitro and in vivo assaysystems. In vitro assays for evaluating therapeutic potential includecell growth assays, soft agar assays and other assays indicative oftumor promoting activity, binding assays capable of determining theextent to which a therapeutic composition will inhibit the binding ofBPC-1 to its coordinate receptor or other binding partner, cell adhesionassays, and the like. For example, antibody to HER2 inhibits binding ofligand to receptor and leads to the inhibition of tumor growth.Additionally, for example, antibodies to EGFR inhibit binding of EGF toreceptor, leading to growth arrest and tumor inhibition. See, also, theExamples below.

Various in vitro assays for determining binding affinity of thetherapeutic composition for its target are also known. For example,binding affinities of BPC-1 antibodies may be determined using a numberof techniques well known in the art (e.g., BIAcore technology). Higheraffinity BPC-1 antibodies are expected to provide greater levels of thedesired inhibition and are therefore preferred.

In vivo, the effect of a BPC-1 therapeutic composition may be evaluatedin a suitable animal model. For example, xenogenic prostate cancermodels wherein human prostate cancer explants or passaged xenografttissues are introduced into immune compromised animals, such as nude orSCID mice, are appropriate in relation to prostate cancer and have beendescribed (Klein et al., 1997, Nature Medicine 3:402-408). For example,POT Patent Application WO 98/16628, Sawyers et al., published Apr. 23,1998, describes various xenograft models of human prostate cancercapable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Various bladder carcinoma modelsare known (see, for example, Russell et al., 1986, Cancer Res.46:2035-2040; Raghavan et al., 1992, Semin. Surg. Oncol. 8:279-284;Rieger et al., 1995, Br. J. Cancer 72:683-690; Oshinsky et al., 1995, J.Urol. 154:1925-1929). Efficacy may be predicted using assays whichmeasure inhibition of tumor formation, tumor regression or metastasis,and the like. See, also, the Examples below.

In vivo assays which qualify the promotion of apoptosis may also beuseful in evaluating potential therapeutic compositions. In oneembodiment, xenografts from bearing mice treated with the therapeuticcomposition may be examined for the presence of apoptotic foci andcompared to untreated control xenograft-bearing mice. The extent towhich apoptotic foci are found in the tumors of the treated mice wouldprovide an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods may be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material which, when combined with the therapeuticcomposition, retains the anti-tumor function of the therapeuticcomposition and is non-reactive with the patient's immune system.Examples include, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Therapeutic formulations may be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition (i.e., BPC-1 monoclonal antibody) in a solutionof preserved bacteriostatic water, sterile unpreserved water, and/ordiluted in polyvinylchloride or polyethylene bags containing 0.9%sterile Sodium Chloride for Injection, USP. The anti-BPC-1 mAbpreparation may be lyophilized and stored as a sterile powder,preferably under vacuum, and then reconstituted in bacteriostatic watercontaining, for example, benzyl alcohol preservative, or in sterilewater prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancerand will generally depend on a number of other factors appreciated inthe art.

Cancer Vaccines

The invention further provides prostate cancer vaccines comprising aBPC-1 protein or fragment thereof. The use of a tumor antigen in avaccine for generating humoral and cell-mediated immunity for use inanti-cancer therapy is well known in the art and has been employed inprostate cancer using human PSMA and rodent PAP immunogens (Hodge etal., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol.159:3113-3117). Such methods can be readily practiced by employing aBPC-1 protein, or fragment thereof, or a BPC-1-encoding nucleic acidmolecule and recombinant vectors capable of expressing and appropriatelypresenting the BPC-1 immunogen.

For example, viral gene delivery systems may be used to deliver aBPC-1-encoding nucleic acid molecule. Various viral gene deliverysystems which can be used in the practice of this aspect of theinvention include, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curro Opin. Immunol. 8:658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a BPC-1 protein or fragment thereof introduced into the patient(e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human BPC-1 cDNA may be employed. In anotherembodiment, BPC-1 nucleic acid molecules encoding specific cytotoxic Tlymphocyte (CTL) epitopes may be employed. CTL epitopes can bedetermined using specific algorithms (e.g., Epimer, Brown University) toidentify peptides within a BPC-1 protein which are capable of optimallybinding to specified HLA alleles.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present BPC-1 antigen to a patient'simmune system. Dendritic cells express MHC class I and II, B7costimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al.,1996, Prostate 29:371-380). Dendritic cells can be used to present BPC-1peptides to T cells in the context of MHC class I and II molecules. Inone embodiment, autologous dendritic cells are pulsed with BPC-1peptides capable of binding to MHC molecules. In another embodiment,dendritic cells are pulsed with the complete BPC-1 protein. Yet anotherembodiment involves engineering the overexpression of the BPC-1 gene indendritic cells using various implementing vectors known in the art,such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25),retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770),lentivirus, adeno-associated virus, DNA transfection (Ribas et al.,1997, Cancer Res. 57:2865-2869), and tumor-derived RNA transfection(Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells expressingBPC-1 may also be engineered to express immune modulators, such asGMCSF, and used as immunizing agents.

Anti-idiotypic anti-BPC-1 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga BPC-1 protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-BPC-1 antibodies that mimic an epitope on aBPC-1 protein (see, for example, Wagner et al., 1997, Hybridoma16:33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al.,1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypicantibody can be used in cancer vaccine strategies.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing BPC-1. Constructs comprising DNA encoding aBPC-1 protein/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take-up the construct and express theencoded BPC-1 protein/immunogen. Expression of the BPC-1 proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against prostate cancer. Variousprophylactic and therapeutic genetic immunization techniques known inthe art may be used.

Kits

For use in the diagnostic and therapeutic applications described orsuggested above, kits are also provided by the invention. Such kits maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe which is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a BPC-1 proteinor a BPC-1 gene or message, respectively. Where the kit utilizes nucleicacid hybridization to detect the target nucleic acid, the kit may alsohave containers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples which follow, none of which are intendedto limit the scope of the invention.

Example 1 Isolation of cDNA Fragment of BPC-1 Gene

Materials and Methods

LAPC Xenografts

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al., 1997, Nature Med. 3:402-408).Androgen dependent and independent LAPC-4 xenografts LAPC-4 (AD and AI,respectively) and LAPC-9 AD xenografts were grown in male SCID mice andwere passaged as small tissue chunks in recipient males. LAPC-4 AIxenografts were derived from LAPC-4 AD tumors. Male mice bearing LAPC-4AD tumors were castrated and maintained for 2-3 months. After the LAPC-4tumors re-grew, the tumors were harvested and passaged in castratedmales or in female SCID mice.

Cell Lines

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 10% fetal calf serum.

RNA Isolation

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer) (SEQ ID NO: 9): 5′TTTTGATCAAGCTT₃₀3′Adaptor 1 (SEQ ID NO: 10):5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ 3′GGCCCGTCCTAG5′Adaptor 2 (SEQ ID NO: 11): 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′3′CGGCTCCTAG5′ PCR primer 1 (SEQ ID NO: 12): 5′CTAATACGACTCACTATAGGGC3′Nested primer (NP)1 (SEQ ID NO: 13): 5′TCGAGCGGCCGCCCGGGCAGGA3′Nested primer (NP)2 (SEQ ID NO: 14): 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes which may be down-regulated in androgenindependent prostate cancer compared to androgen dependent prostatecancer.

Double stranded cDNAs corresponding to the LAPC-4 AD xenograft (tester)and the LAPC-4 AI xenograft (driver) were synthesized from 2 μg ofpoly(A)⁺ RNA isolated from xenograft tissue, as described above, usingCLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotideDPNCDN as primer. First- and second-strand syntheses were carried out asdescribed in the Kit's user manual protocol (CLONTECH Protocol No.PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with DpnII for 3 hrs. at 37° C. Digested cDNA was extracted withphenol/chloroform (1:1) and ethanol precipitated.

Driver cDNA (LAPC-4 AI) was generated by combining in a 1:1 ratio Dpn IIdigested LAPC-4 AI cDNA with a mix of digested cDNAs derived from humanbenign prostatic hyperplasia (BPH), the human cell lines HeLA, 293,A431, Colo205, and mouse liver.

Tester cDNA (LAPC-4 AD) was generated by diluting 1 μl of Dpn IIdigested LAPC-4 AD cDNA (400 ng) in 5 μl of water. The diluted cDNA (2μl, 160 ng) was then ligated to 2 μl of adaptor 1 and adaptor 2 (10 μM),in separate ligation reactions, in a total volume of 10 μl at 16° C.overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation wasterminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) adaptor 1-and adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlayed with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDT A,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec., 66° C.for 30 sec., 72° C. for 1.5 min. Five separate primary PCR reactionswere performed for each experiment. The products were pooled and diluted1:10 with water. For the secondary PCR reaction, 1 μl from the pooledand diluted primary PCR reaction was added to the same reaction mix asused for PCR 1, except that primers NP1 and NP2 (10 μM) were usedinstead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94°C. for 10 sec., 68° C. for 30 sec., 72° C. for 1.5 minutes. The PCRproducts were analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs were generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system.The manufacturer's protocol was used and included an incubation for 50min. at 42° C. with reverse transcriptase followed by RNAse H treatmentat 37° C. for 20 min. After completing the reaction, the volume wasincreased to 200 μl with water prior to normalization. First strandcDNAs from 16 different normal human tissues were obtained fromClontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ IDNO:25) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO:26) to amplifyβ-actin. First strand cDNA (5 μl) was amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCl, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five 1 μl of the PCR reaction was removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: initial denaturation was at 94° C. for 15 sec., followed bya 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min., 72° C. for5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 by β-actinbands from multiple tissues were compared by visual inspection. Dilutionfactors for the first strand cDNAs were calculated to result in equalβ-actin band intensities in all tissues after 22 cycles of PCR. Threerounds of normalization were required to achieve equal band intensitiesin all tissues after 22 cycles of PCR.

To determine expression levels of the 19P1E8 gene, 5 μl of normalizedfirst strand cDNA was analyzed by PCR using 25, 30, and 35 cycles ofamplification using the following primer pairs, which were designed withthe assistance of MIT:

5′-TGC CGT ATG TCA CTG TCT CTA (SEQ ID NO: 15) GGT-3′5′-GAA ATC ATG GGT ATT TCA TGT (SEQ ID NO: 16) GCT-3′These primers were designed from the sequence of the SSH fragment of theinitially isolated 19P1E8 gene. Use of the following primer pair, basedon sequences within the open reading frame of the 19P1 E8 gene, producedthe same expression pattern.

5′-CTC CCA ACT ATC CCA GCA AGT (SEQ ID NO: 17) ATC-3′5′-AAA TCC CAT AGA TTC CAG CTC (SEQ ID NO: 18) TCC-3′

Semi quantitative expression analysis was achieved by comparing the PCRproducts at cycle numbers that give light band intensities.

Results:

Several SSH experiments were conduced as described in the Materials andMethods, supra, and led to the isolation of numerous candidate genefragment clones (SSH clones). All candidate clones were sequenced andsubjected to homology analysis against all sequences in the major publicgene and EST databases in order to provide information on the identityof the corresponding gene and to help guide the decision to analyze aparticular gene for differential expression. In general, gene fragmentswhich had no homology to any known sequence in any of the searcheddatabases, and thus considered to represent novel genes, as well as genefragments showing homology to previously sequenced expressed sequencetags (ESTs), were subjected to differential expression analysis byRT-PCR and/or Northern analysis.

One of the SSH clones, comprising about 700 bp, showed no homology toany known gene or EST sequence was designated 19P1E8. The nucleotidesequence of this SHH clone is shown in FIG. 1, approximately nucleotideresidues 1883-2583. Differential expression analysis by Northern blotshowed that 19P1E8 is expressed in LAPC-4 AD xenograft, and to asignificantly lesser extent in LAPC-4 AI, LAPC-9 AD and LAPC-9 AI (FIG.9). No expression was detected in normal prostate (FIG. 9). Threedistinct transcripts are shown, with sizes of 3.5 kb, 8 kb, and greaterthan 9 kb.

RT-PCR analysis of 19P1E8 expression produced essentially identicalresults (FIG. 5, Panel A). In addition, further RT-PCR expressionanalysis of first strand cDNAs from 16 normal tissues detectedexpression of the 19P1E8 gene only in brain, spleen and testis tissue,and only at very low levels detectable at 35 and not 30 cycles of PCRamplification (FIG. 5, panels B and C). In comparison, substantialexpression was detected in LAPC-4 AD with only 30 cycles (FIG. 5).

Example 2 Isolation of Full Length BPC-1 Encoding cDNA

The 19P1E8 SHH clone above (Example 1) was used to isolate a full length19P1E8 cDNA. Briefly, a cDNA library generated from LAPC-4 mRNA wasscreened with a labeled probe generated from the SSH clone.Specifically, a full length 19P1E8 cDNA of 2639 base pairs (bp) wascloned from an LAPC-4 AD cDNA library generated in lambda ZAP Express(Stratagene).

The cDNA encodes an open reading frame (ORF) of 158 amino acidscontaining a signal sequence and a CUB domain (Complement sub-componentsClr/Cls, Uegf, Bmp 1) (Borck and Beckmann, 1993, J. Mol. Biol.231:539-545). The 5′ UTR (untranslated region) is very GC rich,suggesting that this region contains regulatory elements fortranslation. CUB domains were originally found in complementsub-components Clr and Cis, and were subsequently identified in Uegf(epidermal growth factor related sea urchin protein) and Bmp1 (bonemorphogenetic protein 1), a protease involved in bone development.

In view of the exclusive expression of this gene in brain and itsup-regulation in prostate cancer xenografts, this gene was named BPC-1(Brain/Prostate cancer CUB protein). The nucleotide and deduced aminoacid sequences of the isolated BPC-1 cDNA are shown in FIG. 1. Aschematic representation of the BPC-1 structure is shown in FIG. 2. Anamino acid alignment between the CUB domain of BPC-1 and the CUB domainsof other proteins is shown in FIG. 3. Referring to FIG. 3, of particularinterest is that the CUB domain of BPC-1 is 30-40% identical to the CUBdomains in BMP-1.

The full length BPC-1 cDNA (p19P1E8, clone 6.1) was deposited with theAmerican Type Culture Collection on Aug. 7, 1998 and has been accordedATCC accession number 98833.

Example 3 BPC-1 Gene Expression Analysis—Brain Specific in NormalTissues

Initial analysis of BPC-1 mRNA expression in normal human tissues wasconducted by Northern blotting two multiple tissue blots obtained fromClontech (Palo Alto, Calif.), comprising a total of 16 different normalhuman tissues, using labeled 19P1E8 SSH clone (Example 1) as a probe.RNA samples were quantitatively normalized with a β-actin probe.

The results are shown in FIG. 4. Expression was only detected in normalbrain. The northern blots showed two transcripts of 3.5 kb and 8.0 kb(FIG. 5). The 3.5 kb transcript corresponds to the cDNA identified fromLAPC-4 AD that encodes the BPC-10RF. The larger transcript may encode anun-processed message or an alternative isoform of the gene.

To further explore BPC-1 expression in normal tissues, a multi-tissueRNA dot blot was probed with a BPC-1 probe. Out of 37 different normaltissues tested, only brain regions exhibited detectable levels of BPC-1(FIG. 6). Interestingly, expression of BPC-1 was confined to corticalregions of the brain, such as the temporal, frontal and occipital lobes.Expression was also seen in hippocampus, amygdala, caudate nucleus andputamen. Other brain regions such as thalamus, sub-thalamic nucleus andsubstantia nigra did not express BPC-1. Similarly, no expression wasdetected in other central nervous system (CNS) structures such ascerebellum, spinal cord and medulla oblongata (mid-brain). The RNA dotblot results were confirmed with a northern blot containing RNA fordifferent CNS tissues (FIG. 7).

Example 4 BPC-1 Expression in Fetal Tissues—Broader Expression inDevelopment

CUB domain proteins often are often developmentally regulated. Todetermine if BPC-1 is expressed in human fetal tissue, RT-PCR wasperformed on first strand cDNA derived from 8 different fetal tissues.The results show that BPC-1 is highly expressed in fetal brain, withlower levels detected in all other fetal tissues (FIG. 8). This suggeststhat expression in the adult is exclusive to brain, while expression inother tissues is turned off during development.

Example 5 High Level BPC-1 Expression in Prostate Cancer

To analyze BPC-1 expression in cancer tissues and cell lines, Northernblot analysis was performed on RNA derived from the LAPC prostate cancerxenografts as well as a panel of prostate and bladder cancer cell lines.The results, shown in FIG. 9, reveal the highest levels of BPC-1expression in the LAPC-4 AD prostate cancer xenograft and in the LNCaPprostate cancer cell line, both of which originated from lymph-nodemetastasis of prostate cancer (Klein et al., 1997, Nature Med. 3:402;Horoszewicz et al., 1983, Cancer Res. 43:1809). Lower level expressionof BPC-1 was detected in LAPC-4 AI, LAPC-9 AD and LAPC-9 AI (FIG. 9).Among the bladder cancer cell lines tested, one (5637) showed detectableBPC-1 expression (FIG. 9). No expression was detected in PrEC cells(Clonetics), which represent the basal cell compartment of the prostateand normal prostate.

Example 6 Production of Secreted Recombinant BPC-1 In Vitro

To express recombinant BPC-1 and analyze the subcellular localization ofBPC-1 protein, the full length cDNA was cloned into an expression vectorthat provides a 6H is tag at the carboxyl-terminus (pcDNA 3.1 myc-his,InVitrogen). The construct was transfected into 293T cells which werelabeled for one hour with ³⁵S-methionine. The cells were then washed andincubated in non-radioactive media to chase the labeled proteins forvarious time points. BPC-1-His tagged protein was immunoprecipitatedusing anti-His antibodies (Santa Cruz) from cell extracts and from cellsupernatant (media) at various time points after the chase. Theimmunoprecipitates were analyzed by SDS-PAGE (sodium-dodecyl sulfatepolyacrylamide-gel electrophoresis) with subsequent autoradiography tovisualize ³⁵S-methionine labeled protein.

The results show that BPC-1 protein appears in the cell extract and thecell media immediately after the ³⁵S-methionine labeling period (FIG.10). Within two hours of the chase, nearly all BPC-1 protein is secretedinto the media and remains stable in the media for several hours. Thehalf-life of the protein is estimated to be longer than 24 hours. Vectortransfected cells were also labeled and analyzed using the sameprotocol. Interestingly, a non-specific protein appears in both thevector and the BPC-1 transfected cells. This protein seems to have avery short half-life in the media compared to BPC-1, as it disappearsafter the four hour time point. These results demonstrate that BPC-1 isindeed a secreted protein that appears to be very stable in cell culturemedia.

Example 7 Production of Recombinant BPC-1 Using Baculovirus System

To generate recombinant BPC-1 protein in a baculovirus expressionsystem, BPC-1 cDNA was cloned into the baculovirus transfer vectorpMelBac (Invitrogen) which provides the honeybee mellitin signalsequence for secretion into the media of insect cells. pMelBac-BPC-1 wasco-transfected with helper plasmid pBlueBac4.5 (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus wascollected from cell supernatant and was purified by plaque assay.

Recombinant BPC-1 protein was generated by infection of HighFive insectcells (InVitrogen) with purified baculovirus. Recombinant BPC-1 proteinwas detected in both cell extract and cell supernatant using anti-BPC-1mouse polyclonal antibody (see Example 8, below). Interestingly, thecell extract contains two forms of BPC-1, signal sequence cleaved BPC-1and unprocessed BPC-1 (FIG. 11). The supernatant only contained cleavedmature BPC-1. This recombinant BPC-1 protein may be purified and used invarious cell based assays or as immunogen to generate polyclonal andmonoclonal antibodies specific for BPC-1.

Example 8 Generation of BPC-1 Polyclonal Antibodies

In order to generate antibody reagents that specifically bind to BPC-1,a glutathione-S-transferase (GST) fusion protein encompassing aminoacids 29-93 of the BPC-1 protein was synthesized to serve as immunogen.This fusion protein was generated by PCR-mediated amplification ofnucleotides 877-1,071 (AA 29-93) of the cDNA clone of BPC-1 with thefollowing primers:

(SEQ ID NO: 17) 5′ PRIMER TTGAATTCCAAGCAAACCACCTCAGA EcoRI(SEQ ID NO: 18) 3′ PRIMER AAGCTCGAGTCAGACGGTTCAATAGAGT XhoI

The resultant product was cloned into the EcoR1 and Xhol restrictionsites of the pGEX-2T GST-fusion vector (Pharmacia). RecombinantGST-BPC-1 fusion protein was purified to greater than 90% purity frominduced bacteria by glutathione-sepaharose affinity chromatography.

To generate polyclonal sera to BPC-1, the purified fusion protein wasused as follows. A rabbit was initially immunized with 200 μg ofGST-BPC-1 fusion protein mixed in complete Freund's adjuvant. The rabbitwas injected every two weeks with 200 μg of GST-BPC-1 protein inincomplete Freund's adjuvant. Test bleeds were taken approximately 7-10days following each immunization. ELISA, Western blotting andimmunoprecipitation analyses were used to determine specificity andtiter of the rabbit serum to BPC-1. Cell lines that express BPC-1endogenously, such as LNCaP and cell lines engineered to overexpressBPC-1 by transfection (293T) and by retroviral infection (PC-3 andNIH3T3), were used for characterization of the antiserum. Antiserumrepresenting specific high titer to BPC-1 protein is purified by a 3step process: (1) removal of GST-reactive antibody by depletion over aGST affinity column, (2) BPC-1 specific IgG antibody was isolated bypassage over a GST-BPC-1 affinity column, and (3) protein Gchromatography.

The mouse polyclonal antibody was successfully used for detectingrecombinant BPC-1 expressed in a baculovirus expression system (seeExample 7, above), affinity (nickel) purified MYC/HIS BPC-1 protein(FIG. 13) and recombinant BPC-1 protein in tissue culture supernatantsof cells expressing the BPC-1 gene (FIG. 13). Rabbit polyclonal serumwas also generated and similarly was capable of detecting BPC-1 intissue culture supernatants of cells expressing the BPC-1 gene.

Example 9 Generation of BPC-1 Monoclonal Antibodies

To generate mAbs to BPC-1, 5 Balb C mice were initially immunizedintraperitoneally with 200 μg of GST-BPC-1 fusion protein mixed incomplete Freund's adjuvant. Mice were subsequently immunized every twoweeks with 75 μg of GST-BPC-1 protein mixed in Freund's Incompleteadjuvant for a total of three immunizations. Reactivity of serum fromimmunized mice to full length BPC-1 protein was monitored by ELISA usinga partially purified preparation of HIS-tagged BPC-1 protein expressedfrom 293T cells. Two mice with strongest reactivity were rested forthree weeks and given a final injection of fusion protein in PBS andthen sacrificed four days later. The spleens of the sacrificed mice wereharvested and fused to SPO/2 myeloma cells using standard procedures(Harlow and Lane, 1988). Supernatants from growth wells following HATselection are being screened by ELISA and Western blot to identify BPC-1specific antibody producing clones.

The binding affinity of a BPC-1 monoclonal antibody may be determinedusing standard technology. Affinity measurements quantify the strengthof antibody to epitope binding and may be used to help define whichBPC-1 monoclonal antibodies are preferred for diagnostic or therapeuticuse. The BIAcore system (Uppsala, Sweden) is a preferred method fordetermining binding affinity. The BIAcore system uses surface plasmonresonance (SPR, Welford K., 1991, Opt. Quant. Elect. 23:1; Morton andMyszka, 1998, Methods in Enzymology 295:268) to monitor biomolecularinteractions in real time. BIAcore analysis conveniently generatesassociation rate constants, dissociation rate constants, equilibriumdissociation constants, and affinity constants.

Example 10 Production and Purification of Recombinant BPC-1 Expressed ina Mammalian Expression System

293T cells transiently transfected or 293 cells stably expressing aCMV-driven expression vector encoding BPC-1 with a C-terminal 6×His andMYC tag (pcDNA3.1/mycHIS, Invitrogen) serves as source of secretedsoluble BPC-1 protein for purification (see Example 6, above). TheHIS-tagged BPC-1 protein secreted into the conditioned media is purifiedusing the following method. Conditioned media (500 ml) is concentratedtenfold and simultaneously buffer exchanged into a phosphate buffer (pH8.0) containing 750 mM NaCl and 10 mM imidazole using an amiconultrafiltration unit with a 10 kd MW cutoff membrane. The preparation isthen passed over a nickel metal affinity resin with a 0.5 ml bed volume(Ni-NTA, Qiagen) and washed extensively with phosphate buffer (pH 6.0)containing 10% ethanol and 300 mM NaCl. The HIS-tagged BPC-1 protein isthen eluted with phosphate buffer (pH 6.0) containing 250 mM imidizole.Higher purity preparations are obtained by repeating the abovechromatography step with higher stringency of wash (phosphate buffercontaining 75 mM imidizole) or by passage over an anti-HIS Abimmunoaffinity column. This method was successfully used to purifyrecombinant HIS-BPC-1. A western blot of the purified protein is shownin the far left hand lane of FIG. 13.

Example 11 Retrovirus Mediated Expression of Secreted Human BPC-1

The BPC-1 coding region was subcloned into the retroviral SRαmsvtkneovector (Muller et al., 1991, MCB 11:1785-1792). Retroviruses were madeand used to generate cell lines expressing the BPC-1 gene. The celllines generated are 3T3/BPC-1 and PC3/BPC-1. 3T3 cells acutely infectedwith the SR-αBPC-1 virus express a very high level of BPC-1 mRNA, asdemonstrated by the Northern blot shown in FIG. 12. The PC3/BPC-1 lysateand supernatant were tested for BPC-1 expression by Western blotanalysis using a polyclonal antibody against a GST fusion proteincontaining the N-terminal portion of the BPC-1 protein (aa29-93) asfollows. PC3 and 3T3 cells stably expressing either control (Neo) orBPC-1 encoding retrovirus and 3T3 cells acutely infected with BPC-1retrovirus were cultured in 10 cm tissue culture plates for four days.25 μl of neat supernatant from each line was subjected to Western blotanalysis using a 1:500 dilution of murine anti-BPC-1 polyclonal serum.The blot was then incubated with anti-mouse-HRP conjugated secondaryantibody and BPC-1 specific signals were visualized by enhancedchemiluminescent detection. The results of this Western blot analysisare shown in FIG. 13.

Example 12 BPC-1 Expression Analysis In Vitro and In Vivo

Western and immunoprecipitation analyses of cell lysates and conditionedmedia with BPC-1 specific antibodies may be used to identify andcharacterize BPC-1 protein expression in cell lines and tissues, such asLAPC4 and LAPC9 xenografts, LNCaP prostate cancer cells, 5637 bladdercarcinoma cells, normal human brain lysate, all of which express BPC-1mRNA, as well as a variety of other carcinoma cell lines, xenografts,and normal tissues. Due to the structural homology of BPC-1 to theporcine and bovine spermadhesin family of proteins (Topfer-Petersen etal., Andrologia, 1998), human semen may also contain detectable levelsof BPC-1 protein. Also, given its expression in bladder carcinoma, BPC-1protein may also be detectable in the urine of bladder carcinomapatients. MYC-HIS BPC-1 transfected 293T cells and retrovirallytransduced PC3 and NIH3T3 cells serve as positive controls for BPC-1protein expression (FIG. 13).

Identification and quantitation of BPC-1 protein present in clinicalsamples of human serum, semen, and urine may be carried out by captureELISA as described in the following example. Immunohistochemicalanalysis of BPC-1 protein in normal and cancerous tissues may beconducted on formalin-fixed, paraffin-embedded or frozen tissue sectionsusing standard immunohistochemical methods well known in the art and theBPC-1 antibodies provided herein. Formalin-fixed, paraffin-embeddedsections of LNCaP cells may used as a positive control.

Example 13 BPC-1 Capture ELISA

Capture ELISA may be used to identify and quantify BPC-1 protein presentin clinical samples of human serum, semen, and urine as follows. Thecapture ELISA for BPC-1 is dependent on the generation of at least twomAbs of different isotypes that recognize distinct epitopes of the BPC-1protein or one mAb and a specific rabbit polyclonal serum. One reagentwill serve as the capture (or coating) Ab and the other as the detectionAb. Captured BPC-1 is then visualized by the addition of a secondaryAb-HRP conjugate against the detection antibody followed by incubationwith TMB substrate. Optical density of wells is then measured in aspectrophotometric plate reader at 450 nm. Purified MYC/HIS tagged BPC-1protein serves as a standardization antigen for the ELISA.

Example 14 BPC-1 Expression Results in Anchorage Independent ColonyFormation In Vitro

Retrovirally-infected cells expressing BPC-1 were generated as describedin Example 11 and used along with the respective neo control cell linesto perform soft agar assays to evaluate the oncogenic potential ofBPC-1. The agar assay was performed according to conditions previouslydescribed (Lugo, T. R and O. N. Witte, 1989, Molec. Cell. Biol.9:1263-1270). Briefly, cells were trypsinized and resuspended in Iscovemedium containing 0.3% Noble agar and 20% fetal bovine serum. This cellagar suspension (10⁴ cells/60 mm plate) was plated between a bottom andtop layer of medium containing 0.6% Noble agar and 20% fetal bovineserum. The plates were fed after seven days, and colonies examined andscored two or three weeks after the agar assay was set up, depending onthe size of the colonies. The colonies were counted using a softwarefrom Alphalmager 200. The results are tabulated below in Table 1.

TABLE 1 BPC-1 EXPRESSION INDUCES CELL TRANSFORMATION AVG. NO. COLONIESAVG. NO. COLONIES CELLS [acute infection] [G418 selection for 2 weeks]3T3CL 7/neo 14 65 3T3CL7/BPC-1 116 235

3T3CL7 cells infected with retrovirus expressing BPC-1 or neo were used.Colonies were scored three weeks after the agar assays were set up. The3T3CL7/BPC-1 cells generated about eightfold more colonies compared tothe control plate for acutely infected cells. Using G418 selected cells,there are about 3.6 fold more colonies in the 3T3CL7/BPC-1 platescompared to the 3T3CL7/neo plates.

The above results indicate that the BPC-1 protein induces anchorageindependent growth in cells experimentally engineered to express andsecrete BPC-1 and thus exerts a transforming effect on those cells.

Example 15 BPC-1 Binds to a Cellular Protein

In order to establish whether BPC-1 binds to cellular proteins expressedin prostate cancer cells and other cancer cells or normal cells, twoapproaches were taken. In the first approach, in vitro assay forrecombinant HIS-tagged BPC-1 (Example 6, above) binding to various celllines are used. In another approach, a recombinant alkalinephosphatase-BPC-1 fusion protein are generated using the AP-TAG systemfrom GenHunter Corporation (Nashville, Tenn., ca# Q202), and the AP-TAGfusion used to test BPC-1 binding to a variety of prostate cancer celllines.

A. HIS-Tagged BPC-1 Cell Surface Binding Analysis

PC-3 and NIH3T3 cells are incubated on ice at 4° C. for two hours withconditioned media containing HIS-tagged BPC-1 (from 293T transfectedcells) or media containing purified HIS-tagged BPC-1 or control media.Cells are washed extensively with ice cold PBS with 0.5% FBS and thenincubated with an excess of anti-HIS rabbit polyclonal antibody (5μg/ml, PBS 0.5% FBS) at 4° C. for one hour. Cells are again washed andthen incubated with anti-rabbit FITC conjugated secondary Ab (1:4,000 inPBS/0.5% FBS) for 30 minutes at 4° C. Cell bound BPC-1 is then detectedby fluorimetric analysis of cells in a Cytofluor 4000 fluorimeter (PEBiosystems) and/or by flow cytometry.

As an alternative to the fluorescence-based assay used above, bindingassays can be carried out with ¹²⁵I-labeled BPC-1 protein. Determinationof BPC-1 receptor number and affinity on cells and monitoringinternalization of receptor bound BPC-1 protein is carried out usingstandard published procedures (Raitano and Korc, 1990, J. Biol. Chem.265:10466-10472).

B. Alkaline Phosphatase Tagged BPC-1 Generates Cell Surface Staining inProstate Cancer Cells

Alkaline phosphatase-tagged BPC-1 was generated as follows. The sequenceencoding mature BPC-1 (i.e., without the signal sequence) was clonedinto pAPtag-5 (GenHunter Corp. Nashville, Tenn.). The BPC-1.HindIII andBPC1.BamH1 primers, below, were used to amplify the BPC-1 open readingframe between amino acids 23 and 58 from the plasmid template SRa-19P1E8clone 1. The HindIII and BamH1 digested PCR product was ligated intoHindIII and BgIII digested pAPtag-5 while keeping the IgGK signalsequence, BPC-10RF, and alkaline phosphatase all in frame. The BPC-1-APfusion protein contains an IgGK signal sequence to promote secretionalong with myc/His tags at the carboxy terminus of alkaline phosphatase.

BPC1.HINDIII PRIMER: GTGTAAGCTTCCACCAAGAAAGGAACAGAA (SEQ ID NO: 19)BPC1.BAMHI PRIMER: CACAGGATCCCTTACCAGGTGTGAAATTG (SEQ ID NO: 20)

To detect whether BPC-1 binds with a cell surface receptor on prostatecancer cells, several prostate cancer cell lines and xenograft tissuesare incubated with the BPC-1-AP fusion protein as described (Cheng andFlanagan, 1994, Cell 79:157-168). After washing the cells and adding theAP substrate BCIP, which forms an insoluble blue precipitate upondephosphorylation, BPC-1 receptor binding is determined by identifyingcells staining blue under the light microscope. Various cancer celllines can be examined, including without limitation, various prostatecancer cell lines (e.g., LNCaP, PC-3, DU145, TSUPR, LAPC4) and bladdercarcinoma cell lines. Other cell lines such as PREC prostate cell line,293T, and NIH 3T3, etc. may also be examined. Additionally, the LAPC andother prostate cancer xenografts may be tested.

Equilibrium dissociation rate constants may be calculated to evaluatethe strength of the binding interaction. In addition, the number of cellsurface receptors per cell can be determined. Cell lines or tissues withthe highest binding capacity for BPC-1 would be preferred for cloningthe BPC-1 receptor or other binding partner.

The BPC-1-AP fusion protein was detected in the conditioned media of293T cells transfected with the above construct by Western blotanalysis. Western blot analysis using anti-alkaline phosphatase andanti-HIS antibodies detects the BPC-1-AP fusion protein running atapproximately 90 kDa (FIG. 14).

Conditioned media containing this fusion protein was used to detect a 45kDa binding partner for BPC-1 (FIG. 15), as follows. A western blotprocedure was used to identify a 45 kDa receptor interacting with BPC-1.Lysates from brain, testis, prostate, the xenografts LAPC4AD andLAPC9AD, and the cell lines 3T3, LAPC4, LNCaP, and PC-3 were used togenerate two duplicate western blots. After blocking in 5% milk in PBSfor one hour and washing twice with PBS-Tween for seven minutes each,the blots were incubated with conditioned media from a 293T cell lineproducing only secreted alkaline phosphatase and with media containingBPC-1-AP fusion protein (see FIG. 14). Following three washes withPBS-Tween, the blot was developed using chemiluminescent alkalinephosphatase substrate (Immune-Star, BioRad, cat 170-5010). The resultsare shown in FIG. 15. The arrow (FIG. 15) shows BPC-1-AP binding to a 45kDa protein in 3T3, LAPC9AD, LNCaP, PC-3, and to a lesser extent inLAPC4AD and the LAPC-4 cell line. The 45 kDa protein is not detected inbrain, testis or prostate. The protein interaction is due to BPC-1 andnot AP since the blot shown in FIG. 15 (which was incubated with APconditioned media) did not detect binding the 45 kDa protein.

Example 16 Identification of Potential Signal Transduction Pathways

To determine whether BPC-1 directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing BPC-1 or exposed toexogenously added BPC-1. These transcriptional reporters containconsensus binding sites for known transcription factors which liedownstream of well characterized signal transduction pathways. Thereporters and examples of there associated transcription factors, signaltransduction pathways, and activation stimuli are listed below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

2. SRE-luc, SRFITCF/ELK1; MAPK/SAPK; growth/differentiation

3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

4. ARE-Iuc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

6. CRE-Iuc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

Cells to be assayed for BPC-1-mediated effects include LAPC4, LNCaP,PC3, and NIH3T3. The luciferase reporter plasmids may be introduced bylipid mediated transfection (TFX50, Promega). Luciferase activity, anindicator of relative transcriptional activity, is measured byincubation of cells extracts with luciferin substrate and luminescenceof the reaction is monitored in a luminometer.

Example 17 In Vitro Assays of BPC-1 Function

A. Cell Invasion/Migration/Chemoattraction Assay

Cell lines expressing BPC-1 may be assayed for alteration of invasiveand migratory properties by measuring passage of cells through amatrigel coated porous membrane chamber (Becton Dickinson). Passage ofcells through the membrane to the opposite side is monitored using afluorescent assay (Becton Dickinson Technical Bulletin #428) usingcalcein-Am (Molecular Probes) loaded indicator cells. Cell linesanalyzed include parental and BPC-1 overexpressing PC3, 3T3 and LNCaPcells. To assay whether BPC-1 has chemoattractant properties, parentalindicator cells are monitored for passage through the porous membranetoward a gradient of BPC-1 conditioned media compared to control media.

This assay may also be used to qualify and quantify specificneutralization of the BPC-1 induced effect by candidate cancertherapeutic compositions.

B. Cell Growth Assay

To determine whether BPC-1 alters the growth rate of establishedprostate and nonprostate cell lines, growth curves are generatedcomparing parental cells transduced with a control retroviral vector tocells transduced with a retrovirus encoding the BPC-1 gene. Cell linesto assay include LNCaP, PC3, TsuPR prostate cell lines and murine NIH3T3fibroblasts and various other human non-prostate cell lines. Inaddition, the growth rate of parental cells is assayed in the presenceand absence of exogenously added purified MYC-HIS BPC-1. As analternative source of exogenous BPC-1, conditioned media from therespective BPC-1 retrovirally transduced cell line can be used. Growthof the cell lines is monitored in a 96 well format MTT colorimetricassay (Raitano and Korc, 1990, J. Biol. Chem. 265:10466-10472).

Example 18 In Vivo Models for Studying BPC-1 and Testing Prostate CancerTherapeutic Compositions

A. Determination of Serum BPC-1 Levels in Mice Bearing Xenogenic Tumors

LNCaP prostate cancer cells and LAPC-4 AD xenograft cells express highlevels of BPC-1 as determined by Northern blot analysis. To evaluateBPC-1 as a serum diagnostic marker, SCID mice are injected SQ ororthotopically with either 1×10⁶ LNCaP or LAPC-4 AD cells. Mice areinjected on each flank and tumor growth is monitored by calipermeasurements to reflect length×width×height (L×W×H). The mice are bledat the initial appearance of palpable tumors and every week thereafteruntil tumors are 1,000 mm³ in size. Serial bleeds are screened for thepresence of BPC-1 by an ELISA assay as described above. As a control,serum from the tumor-bearing mice is assessed for the secretion of PSAusing a specific ELISA kit. To confirm BPC-1 expression, tumors areharvested from the mice and screened for BPC-1 expression by Westernblot.

In addition, the 5637 bladder cancer cell line has been shown to expressBPC-1 by Northern blot analysis. To evaluate bladder cancer BPC-1expression, 5637 bladder tumor xenografts are established in SCID miceand serum collected and evaluated for BPC-1 protein by ELISA asdescribed.

Alternatively, prostate cancer cell lines that do not express endogenousBPC-1 and engineered to overexpress BPC-1 may be injected into SCID miceto confirm BPC-1 secretion. These include PC-3, TSUPR1, and DU145.Individual mice are injected SQ with either 1×10⁶ PC3, TSUPR1, and DU145cells expressing an empty tkNeo vector (tkNeo) or a vector containingBPC-1. All mice are injected on each flank and tumor growth is monitoredby caliper measurements as described above. The mice are bled at theinitial appearance of palpable tumors and every week thereafter untiltumors are 1,000 mm³ in size. Differences in tumor growth rate, ifapparent, are noted and studied further (see below). Serial bleeds maybe screened for the presence of BPC-1 by an ELISA assay. To confirmBPC-1 expression, tumors may be harvested from the mice and screened forBPC-1 expression by Western blots.

B. In Vivo Assay for BPC-1 Tumor Growth Promotion

The effect of the BPC-1 protein on tumor cell growth may be evaluated invivo either by gene overexpression or addition of soluble, purifiedBPC-1 protein to tumor-bearing mice. In the first example, SCID mice areinjected SQ on each flank with 1×10⁶ of either PC3, TSUPR1, or DU145cells containing tkNeo empty vector or BPC-1. At least two strategiesmay be used: (1) Constitutive BPC-1 expression under regulation of anLTR promoter, and (2) Regulated expression under control of theecdysone-inducible vector system. Tumor volume is then monitored at theappearance of palpable tumors and followed over time to determine ifBPC-1 expressing cells grow at a faster rate. Additionally, mice may beimplanted with 1×10⁵ of the same cells orthotopically to determine ifBPC-1 has an effect on local growth in the prostate or on the ability ofthe cells to metastasize, specifically to lungs, lymph nodes, and bonemarrow.

In the second example, purified BPC-1 protein is be evaluated for aneffect on tumor cell growth in vivo. Mice are first divided into groupsinjected SQ with either 1×10⁶ LNCaP or LAPC-4 AD cells, which expressBPC-1, or PC3 cells, which do not express BPC-1. On the same day astumor cells are injected, groups are injected IV with a range ofpurified BPC-1 protein (for example 100, 500, or 1,000 μg). As acontrol, one group of each tumor type is injected with PBS only.Injections continue two times per week for four consecutive weeks untiltumors grow and reach a size of 1,000 mm³. Tumor volume is followed todetermine if BPC-1 has a dose response effect on tumor growth.

In a separate set of experiments to determine if BPC-1 accelerates tumorgrowth, LNCaP, LAPC-4 AD, and PC3 tumors may be allowed to establish SQto a size of 100 mm³, at which time purified BPC-1 protein is injectedIV in the doses and regimen indicated above. To determine if BPC-1promotes metastasis, the same tumors may also be implantedorthotopically, and, after tumors have been established (determined bycirculating PSA levels), purified BPC-1 can be administered as describedand metastatic growth evaluated.

The above assays are also useful to determine the BPC-1 inhibitoryeffect of candidate therapeutic compositions, such as for example, BPC-1antibodies and intrabodies, BPC-1 mRNA antisense molecules andribozymes, and BPC-1 receptor compositions.

C. In Vivo BPC-1 Antibody Tumor Inhibition Assay

To study the effect of BPC-1 specific mAbs on the formation and growthof tumors, mice are divided in groups of either BPC-1 positive LNCaP,LAPC-4 AD, and PC3-BPC-1 tumors, or PC3-tkNeo, which does not expressBPC-1. To evaluate an effect on tumor formation, mice are injected with1×10⁶ tumor cells SQ and on the same day are injected IP with a range ofBPC-1 specific mAb or control Ig (for example, 100, 500, or 1,000 μg).Injections of mAbs continue 2 times per week for 4 consecutive weeks.Tumor growth is followed as described above. Alternatively, to evaluatean effect on established tumors, mice are divided into groups bearingestablished tumors 100 mm³ in size and are injected IP with mAbsaccording to the doses and regimen described previously. Tumor volume isfollowed to determine the mAb's effect on growth of established tumors.

To study effect on metastasis, 1×10⁶ of LAPC-4 AD cells are injectedorthotopically into SCID mice. At the same time, the mice are injectedIP with a range of anti-BPC-1 mAb or control Ig as described above.Tumor growth is followed by weekly determinations of circulating PSA. Atthe end of the antibody administration, the mice are sacrificed andlocal tumor growth and metastasis to lungs, lymph nodes, and bone marroware evaluated. To examine an effect on mice with established tumors,LAPC-4 AD are injected orthotopically and PSA levels are followedweekly. When PSA reaches measurable levels, the mice are injected withthe same dose and regimen of mAbs described. The mice are sacrificedafter the completion of antibody injections to evaluate local tumorgrowth as well as metastasis.

Example 19 Molecular Cloning of the BPC-1 Receptor or Binding Partner

Expression cloning strategies such as described in Tartaglia et al.,1995, Cell 83:1263-1271 and Cheng and Flanagan, 1994, Cell 79:157-168and others may be used to clone the receptor for BPC-1. An expressionlibrary is first constructed from cells showing BPC-1-AP binding. Thelibrary may be constructed in pools of approximately 1,000 clones andthen screened by a sib selection procedure. Transient transfection ofCOS cells with DNA from each pool and subsequent screening with BPC-1-APbinding, washing, and staining for AP activity identifies cells bindingBPC-1 and consequently expression of BPC-1 receptor. After successiverounds of pool subdivision and screening, single colonies binding toBPC-1-AP can be identified.

An alternative approach to cloning BPC-1 receptor/binding partner genesutilizes expression cloning in phage (Stone, J. in Current Protocols inMolecular Biology (1997):20.3.1-20.3.9). For example, a LAPC-9 AD phageexpression library in Lambda Zap Express (Stratagene) may be used.Membrane lifts can be probed using BPC-1-AP and positive clones detectedwith an alkaline phosphatase chemiluminescent reagent (e.g., BioRad).Plaques binding BPC-1-AP and producing a blue precipitate are selectedand plasmids isolated and evaluated for the receptor/binding partnersequences. This approach may also result in the identification ofcytoplasmic or secreted proteins interacting with BPC-1.

Throughout this application, various publications are referenced withinparentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

1. A purified polypeptide, comprising the amino acid sequence of SEQ IDNO: 2.